Glutamat og GABA: Hovedaktører i nevronal metabolisme cand.med. Elisabeth Olstad Ovennevnte avhandling er funnet verdig til å forsvares offentlig for graden PhD i nevrovitenskap. Disputasen finner sted i Auditoriet, Laboratoriesenteret, St. Olavs Hospital fredag 02. mars 2007 kl. 12.15
146
Embed
Glutamat og GABA: Hovedaktører i nevronal metabolisme · Glutamat og GABA: Hovedaktører i nevronal metabolisme cand.med. Elisabeth Olstad Ovennevnte avhandling er funnet verdig
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Glutamat og GABA:
Hovedaktører i nevronal metabolisme
cand.med.
Elisabeth Olstad
Ovennevnte avhandling er funnet verdig til
å forsvares offentlig for graden PhD i nevrovitenskap.
Disputasen finner sted i Auditoriet, Laboratoriesenteret, St. Olavs Hospital
fredag 02. mars 2007 kl. 12.15
Glutamate and GABA: Major Players in Neuronal Metabolism
i
PREFACE AND ACKNOWLEDGEMENTS
This thesis presents experimental work carried out from June
2001 until August 2006 at the Norwegian University of Science and
Technology (NTNU). The studies have been done at the Department of
Neuroscience at The Faculty of Medicine and was supported by NTNU,
the Central Norway Regional Health Authority (Helse Midt-Norge)/ St.
Olavs Hospital, Trondheim University Hospital and the Norwegian
Research Council. An initial two month student summer grant inspired
me to pursue the medical student research program (forskerlinjen). I
was the first female to graduate from this program in June of 2005,
when I also finished my medical degree. Finally, in August 2005, I
officially started on my PhD. My supervisors have been Professor
Ursula Sonnewald and from August 2005 also dr.scient. Hong Qu. I
am grateful for all their help and enthusiasm! I also wish to thank my
other co-authors for important contributions to this research,
especially Professor Arne Schousboe and Associate Professor Helle
Waagepetersen at the Danish University of Pharmaceutical Sciences.
I also have to thank Bente Urfjell and Lars Evje for technical
assistance. Bente has taught me all I know about practical laboratory
work, her help and friendship has been and still is, greatly
appreciated! I would also like to thank the rest of the research group,
especially PhD student Torun Melø and post doctoral Øystein Risa;
they have made me a little wiser when it comes to MRS. Dr.ing. Turid
Nilsen’s help with GC/MS has been invaluable and deserves special
thanks! For coffee breaks and friendship, thanks also to Silje, Elvar
and Eiliv!
Finally, I want to thank Eivind, and also my parents and my
sister for their patience, love and support! Thank you for believing in
me!
Glutamate and GABA: Major Players in Neuronal Metabolism
ii
SUMMARY
Disturbance of neuronal metabolism has implications for a
number of neurological and psychiatric conditions, and enhanced
knowledge of this is important in developing new methods for treating
such disorders. The present research was undertaken to aid
understanding of diseases related to disturbance in glutamate and
γ-amino butyric acid (GABA) metabolism.
Two different types of neuronal cell cultures were used in these
studies; one containing GABAergic neurons of cerebral neocortical
origin and one containing cerebellar neurons. The latter consists
primarily of glutamatergic granule neurons in addition to ~6 %
GABAergic neurons and a small number of astrocytes. Metabolism was
studied by 13C magnetic resonance spectroscopy (MRS) and mass
spectrometry (MS) after adding 13C-labeled precursors
([1-13C]glucose, [U-13C]glutamate or [U-13C]glutamine) to the
medium of these cultures. High performance liquid chromatography
(HPLC) was used to quantify different amino acids in cell extracts and
medium. The amount of protein in the cultures was determined to
assess cell damage.
In the cerebellar neuronal cultures, GABA was present in
surprisingly large amounts compared to neocortical GABAergic
cultures. 13C MRS experiments showed that GABA was actively
synthesized throughout the culture period by the subpopulation of
glutamate decarboxylase (GAD) positive (GABAergic) neurons and
subsequently distributed to the other cells in the culture, i.e. to the
granule neurons. The function of GABA in these glutamatergic neurons
still remains uncertain; however, roles as neurotrophic and
neuroprotective agent as well as substrate for energy production have
been suggested.
Glutamate and GABA: Major Players in Neuronal Metabolism
iii
As shown previously, both glutamate and glutamine were
shown to be excellent precursors for intermediary metabolism in
cerebellar neurons. However, it was concluded that glutamate was
preferred over glutamine, suggesting that these neurons rely more on
reuptake of released glutamate than of supply of glutamine from
astrocytes for glutamate homeostasis. This is not surprising when
considering the cerebellar structure, with few astrocytes compared to
neurons and a relatively large distance between astrocyte and
synapse.
Exposure of cerebellar cultures to 50 μM kainic acid (KA), a
potent glutamate agonist, which is known to eliminate vesicular
release of GABA in these cultures, only marginally affected glutamate
and GABA metabolism, whereas increasing the KA concentration to
0.5 mM led to a reduction of both GABA and glutamate metabolism
compared to unexposed cultures. It was previously believed that
treatment with 50 μM KA eliminated the GABAergic neurons in
cerebellar cultures, and KA has therefore been added in order to
obtain essentially pure glutamatergic granule cell cultures. Although
KA treatment abolishes vesicular GABA release, the GABA
synthesizing cells are not eliminated by this treatment and still
produce GABA in substantial amounts.
Results from the present studies can only be understood in
terms of inter- and intracellular compartmentation of metabolism. The
main focus of metabolic compartmentation studies has been on the
two compartments made up by neurons and astrocytes. One pathway
previously believed to take place in the astrocytic but not in the
neuronal compartment, is the pyruvate recycling pathway for
complete tricarboxylic acid (TCA) cycle oxidation of glutamate.
Despite this, in one of the present studies, such recycling was clearly
present in both astrocytic and neuronal cultures from cerebellum.
Glutamate and GABA: Major Players in Neuronal Metabolism
iv
LIST OF PAPERS
This thesis is based on the following publications:
Paper 1
Sonnewald U, Olstad E, Qu H, Babot Z, Cristòfol R, Suñol C,
Schousboe A and Waagepetersen H. First direct demonstration of
extensive GABA synthesis in mouse cerebellar neuronal cultures. J
Neurochem (2004) 91, 796-803
Paper 2
Sonnewald U, Kortner TM, Qu H, Olstad E, Suñol C, Bak LK,
Schousboe A and Waagepetersen HS. Demonstration of extensive
GABA synthesis in the small population of GAD positive neurons in
cerebellar cultures by the use of pharmacological tools. Neurochem
Int (2006) 48, 572-578
Paper 3
Olstad E, Qu H and Sonnewald U. Glutamate is preferred over
glutamine for intermediary metabolism in cultured cerebellar neurons.
J Cereb Blood Flow Metab (2006) in press
Paper 4
Olstad E, Qu H and Sonnewald U. Long-term kainic acid exposure
reveals compartmentation of glutamate and glutamine metabolism in
cultured cerebellar neurons. Neurochem Int (2006) in press
Paper 5
Olstad E, Olsen GM, Qu H and Sonnewald U. Pyruvate recycling in
cultured neurons from cerebellum. J Neurosci Res (2006) in press
Glutamate and GABA: Major Players in Neuronal Metabolism
v
ABBREVIATIONS
acetyl CoA acetyl coenzyme A AMPA α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid AOAA aminooxyacetic acid ATP adenosine triphosphate CNS central nervous system DMEM Dulbecco’s minimum essential medium EAAT excitatory amino acid transporter FCS fetal calf serum GABA γ-amino-butyric acid GABA-T GABA aminotransferase GAD glutamate decarboxylase GAT GABA transporter GC gas chromatography GDH glutamate dehydrogenase GLUT glucose transporter GS glutamine synthetase GSH glutathione GVG γ-vinyl GABA HPLC high performance liquid chromatography KA kainic acid MR magnetic resonance MRS magnetic resonance spectroscopy MS mass spectrometry NMDA N-methyl-D-aspartate nOe nuclear Overhauser effect OAA oxaloacetate OPA o-phthaldialdehyde PAG phosphate activated glutaminase PC pyruvate carboxylase TCA tricarboxylic acid
Glutamate and GABA: Major Players in Neuronal Metabolism
vi
TABLE OF CONTENTS
Preface and Acknowledgements i
Summary ii
List of Papers iv
Abbreviations v
Table of Contents vi
1 INTRODUCTION 1
1.1 Medical Aspects of Neuronal Metabolism 1
1.2 The Cells of the Brain 5
1.2.1 Neurons and Neurotransmission 6
1.2.2 Glia 7
1.2.3 Neuronal-Glial Interaction and Compartmentation 8
1.3 Transport and Metabolism of Glucose, Glutamate
and GABA 10
1.3.1 Glucose 10
1.3.2 Glutamate 11
1.3.3 GABA 15
2 OBJECTIVES 19
3 METHODS 21
3.1 Neuronal Cell Cultures 21
3.2 Identification of Metabolites and Metabolic Pathways
by MRS 24
3.2.1 MRS in Neurobiological Research 24
3.2.2 Basic MR Theory 24
3.2.3 13C MRS 27
Glutamate and GABA: Major Players in Neuronal Metabolism
vii
3.3 Mass Spectrometry 30
3.3.1 Detection of 13C Labeling in Metabolites by MS 30
3.3.2 Basic GC/MS Theory 31
3.4 13C Labeling Patterns 34
3.4.1 Labeling from [1-13C]glucose 34
3.4.2 Labeling from [U-13C]glutamate and
[U-13C]glutamine 36
3.5 Identification and Quantification of Amino Acids
by HPLC 39
3.6 Protein Quantification 40
4 SUMMARY OF PAPERS 41
5 DISCUSSION 47
6 CONCLUSIONS 59
List of References ix
Paper 1
Paper 2
Paper 3
Paper 4
Paper 5
Glutamate and GABA: Major Players in Neuronal Metabolism
viii
Glutamate and GABA: Major Players in Neuronal Metabolism
1
1 INTRODUCTION
1.1 Medical Aspects of Neuronal Metabolism
Normal energy metabolism in the brain has several unusual
features compared to other organs, and disturbance of this
metabolism is considered important in many brain disorders (Balázs et
al., 2006). One of the features of normal brain function is the high
metabolic rate; in fact, the brain is one of the most metabolically
active organs in mammals, illustrated by the fact that despite
constituting modest 2 % of the total body mass, the brain accounts
for an astounding 20 % of the resting body’s oxygen consumption
(McKenna et al., 2006a). This oxygen is almost exclusively used for
oxidation of glucose (Sokoloff, 1960), the main energy source of the
brain. Under extraordinary conditions, like prolonged starvation, the
mature brain can adapt to using ketone bodies produced in the liver
from fat to cover some of the energy needs (Stryer, 1995b).
Nevertheless, the brain is not very flexible when it comes to energy
substrates compared to other organs and is critically dependent on
aerobic metabolism of glucose (Dugan and Kim-Han, 2006;
McKenna et al., 2006a). Another feature is the limited intrinsic
energy stores of the brain. Although some glycogen can be stored,
mainly in astrocytes (Pfeiffer-Guglielmi et al., 2003; McKenna et al.,
2006a), the brain has no significant energy reserve. It has been
estimated that if glycogen was the only source of fuel, it would be
consumed in a few minutes (McKenna et al., 2006a). Thus, the brain
is dependent on a constant supply of glucose and oxygen via the
blood.
The dependence of a constant blood supply carrying glucose
and oxygen makes the brain particularly vulnerable to ischemic injury
Glutamate and GABA: Major Players in Neuronal Metabolism
2
(Dugan and Kim-Han, 2006). This is most often seen as a disruption
of blood supply to a part of the brain caused by a thromboembolic
occlusion of an intracranial artery, commonly known as a stroke
(Smith, 2004). This is the most common neurological disorder in
terms of both morbidity and mortality (De Girolami et al., 1999).
When the blood flow, and thereby the energy supply, to the brain is
impaired, ATP levels decreases, which in turn affects the active ion
pumps, such as the Na+/K+ ATPase. The ion gradients over the cell
membrane, and thus the membrane potential will be disrupted, and
the neurons are depolarized (Smith, 2004; Balázs et al., 2006; Dugan
and Kim-Han, 2006). This causes a cascade of events ultimately
leading to cell death. With the reduction of cerebral blood flow in
ischemia, the extracellular glutamate concentration is substantially
elevated (Smith, 2004). This leads to excessive activation of
excitatory amino acid receptors, in particular glutamate receptors,
causing cell death, a mechanism referred to as excitotoxicity (Olney,
1978).
A role for excitotoxicity has been implicated in the etiology of
many neurodegenerative diseases, including Alzheimer’s disease,
Parkinson’s disease and amyotrophic lateral sclerosis (ALS) (Mattson,
2003; Balázs et al., 2006). Excessive or prolonged activation of
specific glutamate receptors results in a rise in intracellular Ca2+
concentration, triggering a cascade of intracellular events culminating
in neurodegeneration. Different types of neurons have different
vulnerability to excitotoxicity, depending on their receptors, Ca2+
permeability and ability to handle an increase in intracellular Ca2+
(Balázs et al., 2006). The glutamatergic N-methyl-D-aspartate
(NMDA) receptors are the primary receptors activating excitotoxicity
because of their high permeability to Ca2+, although other glutamate
receptors can initiate excitotoxicity by allowing excessive Ca2+ entry.
Studies have shown that cytoplasmic Ca2+ is insufficient to cause
Glutamate and GABA: Major Players in Neuronal Metabolism
3
neuronal death in itself, and that mitochondrial Ca2+ accumulation is
essential for excitotoxic cell death (Stout et al., 1998; Nicholls et al.,
2003). Ca2+ causes mitochondria to generate reactive oxygen species,
and this oxidative damage can initiate cell death. Diseases such as
Alzheimer’s disease, Parkinson’s disease and ALS are accompanied by
increased oxidative stress, and in these patients, neurons are more
susceptible to excitotoxic death (Balázs et al., 2006). Thus,
excitotoxicity contributes to oxidative stress, which in turn reduces
the threshold for excitotoxicity, leaving cells more vulnerable to
injury. This is one of the reasons why excitotoxicity contributes to
many neurodegenerative diseases. Knowledge of regulation of
glutamate receptors in Alzheimer’s disease, Parkinson’s disease and
ALS have resulted in clinically efficacious drugs and new therapeutic
medications are continually being developed (Mattson, 2003).
Another common neurological disorder is epilepsy,
characterized by recurrent, spontaneously occurring seizures with
symptoms caused by abnormal excessive or hypersynchronous
neuronal activity in the brain (Blume et al., 2001; Fisher et al., 2005).
The epileptic seizure is a pathophysiological process characterized by
a synchronous activation of a large group of neurons in the brain. This
may be caused by a disturbance in the fine-tuned balance between
excitatory glutamatergic and inhibitory GABAergic neurotransmission,
a theory supported by the fact that inhibition of γ-amino butyric acid
(GABA) synthesis and administration of GABA antagonists and
1% t-BDMS-Cl (tert-butyldimethylchlorosilane) as described by
Mawhinney et al. (1986). The cell extract sample is then injected into
the injection port of the GC, where it is immediately vaporized and
carried to the column by the carrier gas. It is important that the
carrier gas is inert and does not react with the sample or column, and
for this reason helium was used in the present studies. The column
used was a capillary column coated with silica (Varian WCOT fused
silica 25 m x 0.25 mm ID coating CP-Sil 5CB-MS). The various
components in the cell extract sample travel through the column at
different speeds based on their chemical and physical characteristics
(mass, shape, interaction with column surface, etc.), and they are
separated. Each component ideally produces a specific peak which
appears in the chromatogram after a characteristic retention time.
After separation of the different metabolites in the cell extracts
by GC, MS is used to separate molecules of the same metabolite with
different masses (M, M+1, M+2, etc.), i.e. different isotopomers of
each metabolite. The gas carrying the separated metabolites is let into
the ionization chamber where a beam of electrons is accelerated with
a high voltage. The molecules in the sample are shattered into ionized
Glutamate and GABA: Major Players in Neuronal Metabolism
32
fragments upon collision with the high voltage electrons. The charged
fragments are electrically focused into an intense ion beam which
enters the quadrupole analyzer. The electrically charged poles of the
quadrupole create an electromagnetic field, and the ion beam is
forced into a corkscrew, three-dimensional sine wave. Across the
quadrupole rods a combined field of direct current and an oscillating
radio frequency signal is applied. This interrupts the paths of all ions
except for those with one specific mass to charge ratio. A mass
spectrum is obtained by scanning through the mass range of interest
over time. When using the instrument’s SCAN mode, the whole mass
range is scanned. However, when knowing which masses to look for,
the instrument is set to scan over a very small mass range, the
selected ion monitoring (SIM) mode. The narrower the mass range
the more specific the SIM assay. The method used in the present
studies was developed using the SCAN mode for analyzing standard
solutions of individual compounds to determine the retention time and
the masses of interest for the compounds. When this was done, a SIM
method was set up with retention time windows in which the
instrument was set to scan over a few masses in order to enhance
sensitivity. After being selected in the quadrupole, the charged
particles travel in a curved path towards the detector, and on the way
the charge is amplified through collisions with the detector surface.
The computer linked to the GC/MS instrument gives a plot of
relative abundance against the mass to charge ratio value of the ions.
An example of two gas chromatograms and mass spectra is shown in
Figure 3.5. The peaks are integrated and the percentage of mono-,
double-, triple labeling etc. in a compound is calculated after
correction for natural abundance determined in a standard solution of
unlabeled compounds. However, as mentioned earlier, this method
does not differentiate between isotopomers containing the same
number of 13C atoms in different positions.
Glutamate and GABA: Major Players in Neuronal Metabolism
33
FIGURE 3.5 Parts of gas chromatograms (top) and mass spectra (bottom) from a
standard solution of unlabeled compounds (left) and a sample of cell extract from
cerebellar neuronal cultures incubated for two hours in medium containing 0.25 mM [U-13C]glutamate, for details see paper 3. The chromatograms show the malate, aspartate
and glutamate peaks, and the mass spectra show masses M (unlabeled) to M+5
(uniformly labeled) for glutamate.
Glutamate and GABA: Major Players in Neuronal Metabolism
34
3.4 13C Labeling Patterns
Understanding the labeling patterns from 13C labeled
precursors involves knowledge about cell metabolism. This can be
found in a biochemistry textbook, for example the one written by
Stryer (1995a)
3.4.1 Labeling from [1-13C]glucose
In papers 1 and 2, neuronal cell cultures prepared for MRS
analysis were cultured in medium containing [1-13C]glucose for the
whole culture period. Glucose is the most important substrate for
neuronal metabolism, and the metabolites made from this labeled
glucose, will contain 13C and thus be detectable by 13C MRS. In order
to interpret the MR-spectra and understand the results obtained from
these spectra, it is necessary to know the relevant metabolic
conversions of [1-13C]glucose. This is illustrated in Figure 3.6.
FIGURE 3.6 Metabolism of [1-13C]glucose in neurons. ● represents 13C and ○ represents 12C atoms. PDH is the enzyme pyruvate dehydrogenase which catalyzes the reaction
from pyruvate to acetyl-CoA. *Unlabeled pyruvate will have the same conversions as [3-13C]pyruvate, but the products will not be detectable by 13C MRS.
Glutamate and GABA: Major Players in Neuronal Metabolism
35
Through glycolysis, [1-13C]glucose is converted to two
pyruvate molecules. One of them will contain a 13C-atom in the third
position ([3-13C]pyruvate), whereas the other one will contain only 12C-atoms (the natural abundance of 13C of 1.1 % is not taken into
consideration). [3-13C]pyruvate can be converted to [3-13C]lactate or
[3-13C]alanine. Alternatively, [3-13C]pyruvate may enter the
tricarboxylic acid cycle via pyruvate dehydrogenase (PDH) as [2-13C]acetyl-CoA. In the TCA cycle, [2-13C]acetyl-CoA is combined with
oxaloacetate (OAA) and converted through several steps to α-
ketoglutarate with 13C-labeling in the C-4 position, which may leave
the TCA cycle and form [4-13C]glutamate, which in turn can be
converted to [2-13C]GABA.
If α-[4-13C]ketoglutarate does not leave the cycle, it will (after
several steps) appear as [2-13C]oxaloacetate (OAA) or [3-13C]oxaloacetate (because succinate, one of the intermediate
compounds between α-ketoglutarate and OAA in the TCA cycle, is a
symmetrical molecule). 13C-labeled OAA can be converted to [2-13C]aspartate or [3-13C]aspartate by transamination, or condense with
a new acetyl-CoA-molecule, labeled or unlabeled with 13C (from
labeled or unlabeled pyruvate), and make a second turn in the TCA
cycle. If 13C-labeled OAA reacts with unlabeled acetyl-CoA, the
resulting labeling (after several steps) in glutamate and GABA is [2-13C]- and [3-13C]glutamate and [3-13C]- and [4-13C]GABA. If 13C-
labeled OAA reacts with [2-13C]acetyl-CoA, [2,4-13C]- and [3,4-13C]glutamate and [2,4-13C]- and [2,3-13C]GABA are formed. The
labeling [1-13C]glucose in glutamate and GABA after one and two
turns in the TCA cycle is shown in Figure 3.7.
After more turns in the TCA cycle and reactions between
molecules with and without 13C-atoms in different positions, the
possibilities are many for 13C-labeling of the different metabolites, and
Glutamate and GABA: Major Players in Neuronal Metabolism
36
the picture becomes more complicated than shown in Figures 3.6 and
3.7.
FIGURE 3.7 Labeling of 13C in glutamate and GABA from [1-13C]glucose in the first and
second turn of the TCA cycle. ● represents 13C and ○ represents 12C atoms.
3.4.2 Labeling from [U-13C]glutamate and [U-13C]glutamine
In papers 3, 4 and 5, neuronal cell cultures from cerebellum
were incubated in medium containing [U-13C]glutamate or [U-13C]glutamine. When taken up by the neurons, the latter can be
converted into the former, and from here on the labeling patterns are
the same for the two precursors. [U-13C]glutamate can together with
cysteine and glycine form the tripeptide glutathione (GSH). The
labeled glutamate incorporated in glutathione can be identified by 13C
MRS; its peaks will appear in a different location in the spectrum than
free glutamate (Figure 3.3). Another possibility for [U-13C]glutamate
Glutamate and GABA: Major Players in Neuronal Metabolism
37
is conversion into [U-13C]GABA catalyzed by the enzyme GAD. [U-13C]GABA could not be detected in cell extracts by MRS after two
hours incubation in medium containing [U-13C]glutamate. However,
using MS, the M+4 isotopomer of GABA (representing [U-13C]GABA)
was detected. A third option for [U-13C]glutamate is the formation of
α-[U-13C]ketoglutarate, which is metabolized in the TCA cycle. After
several steps in the TCA cycle, labeled α-ketoglutarate is turned into
[U-13C]oxaloacetate, which can condense with unlabeled acetyl CoA to
form [3,4,5,6-13C]citrate. The resulting glutamate isotopomer (after
several steps) is [1,2,3-13C]glutamate formed from α-[1,2,3-13C]ketoglutarate. This first turn for [U-13C]glutamate in the TCA cycle
is shown in Figure 3.8.
FIGURE 3.8 Metabolism of [U-13C]glutamate in neurons. ● represents 13C and ○
represents 12C atoms. Glutathione is a tripeptide, and the black box is representing
labeled glutamate, while cysteine and glycine are amino acids without 13C labeling,
represented by white boxes.
Glutamate and GABA: Major Players in Neuronal Metabolism
38
If α-[1,2,3-13C]ketoglutarate does not leave the TCA cycle as
[1,2,3-13C]glutamate, but continues its voyage in the cycle, 13C
labeling is distributed amongst the TCA cycle intermediates as
presented in Figure 3.9.
FIGURE 3.9 Schematic representation of possible isotopomers of metabolites arising
from [U-13C]glutamate or [U-13C]glutamine via the three first turns in the TCA cycle in
neurons: ● represents 13C and ○ represents 12C atoms. For clarity, the labeling of
fumarate, malate, OAA and isocitrate is left out; the three first compounds are labeled
in the same manner as succinate and the latter as citrate, although the numbering of
the C atoms differs. GLU: glutamate; αKG: α-ketoglutarate; SUC-CoA: succinyl-CoA;
Westergaard N, Fosmark H, Schousboe A (1991) Metabolism and release of glutamate
in cerebellar granule cells cocultured with astrocytes from cerebellum or cerebral
cortex. J Neurochem 56:59-66.
Westergaard N, Sonnewald U, Petersen SB, Schousboe A (1995) Glutamate and
glutamine metabolism in cultured GABAergic neurons studied by 13C NMR spectroscopy
may indicate compartmentation and mitochondrial heterogeneity. Neurosci Lett 185:24-
28.
White LE, Hodges HD, Carnes KM, Price JL, Dubinsky JM (1994) Colocalization of
excitatory and inhibitory neurotransmitter markers in striatal projection neurons in the
rat. J Comp Neurol 339:328-340.
Wu JY, Roberts E (1974) Properties of brain L-glutamate decarboxylase: inhibition
studies. J Neurochem 23:759-767.
Yu AC, Drejer J, Hertz L, Schousboe A (1983) Pyruvate carboxylase activity in primary
cultures of astrocytes and neurons. J Neurochem 41:1484-1487.
Yu AC, Hertz E, Hertz L (1984) Alterations in uptake and release rates for GABA,
glutamate, and glutamine during biochemical maturation of highly purified cultures of
cerebral cortical neurons, a GABAergic preparation. J Neurochem 42:951-960.
Glutamate and GABA: Major Players in Neuronal Metabolism
xxii
Paper 1
First direct demonstration of extensive GABA synthesis in
mouse cerebellar neuronal cultures
Sonnewald U, Olstad E, Qu H, Babot Z, Cristòfol R, Suñol C, Schousboe A
and Waagepetersen H.
J Neurochem (2004) 91, 796-803
Paper I is not included due to copyright.
Paper 2
Demonstration of extensive GABA synthesis in the small
population of GAD positive neurons in cerebellar cultures by the
use of pharmacological tools
Sonnewald U, Kortner TM, Qu H, Olstad E, Suñol C, Bak LK, Schousboe A
and Waagepetersen HS.
Neurochem Int (2006) 48, 572-578
Demonstration of extensive GABA synthesis in the small
population of GAD positive neurons in cerebellar cultures
by the use of pharmacological tools
Ursula Sonnewald a,*, Trond M. Kortner a, Hong Qu a, Elisabeth Olstad a, Cristina Sunol b,Lasse K. Bak c, Arne Schousboe c, Helle S. Waagepetersen c
a Department of Neuroscience, Faculty of Medicine, Norwegian University of Science and Technology (NTNU),
Olav Kyrres Gate 3, N-7489 Trondheim, Norwayb Department of Neurochemistry, Institut d’Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientıficas,
CSIC, IDIBAPS, E-08036 Barcelona, Spainc Department of Pharmacology and Pharmacotherapy, Danish University of Pharmaceutical Sciences, DK-2100 Copenhagen, Denmark
Received 19 October 2005; received in revised form 16 January 2006; accepted 17 January 2006
Available online 3 March 2006
www.elsevier.com/locate/neuint
Neurochemistry International 48 (2006) 572–578
Abstract
Cultures of dissociated cerebella from 7-day-old mice were maintained in vitro for 1–13 days. GABA biosynthesis and degradation were studied
during development in culture and pharmacological agents were used to identify the enzymes involved. The amount of GABA increased, whereas
that of glutamate was unchanged during the first 5 days and both decreased thereafter. The presence of aminooxyacetic acid (AOAA, 10 mM) which
inhibits transaminases and other pyridoxal phosphate dependent enzymes including GABA-transaminase (GABA-T), in the culture medium
caused an increase in the intracellular amount of GABA and a decrease in glutamate. The GABA content was also increased following exposure to
the specific GABA-T inhibitor g-vinyl GABA. From day 6 in culture (day 4 when cultured in the presence of AOAA) GABA levels in the medium
were increased compared to that in medium from 1-day-old cultures. Synthesis of GABA during the first 3 days was demonstrated by the finding
that incubation with either [1-13C]glucose or [U-13C]glutamine led to formation of labeled GABA. Synthesis of GABA after 1 week in culture,
when the enzymatic machinery is considered to be at a more differentiated level, was shown by labeling from [U-13C]glutamine added on day 7.
Altogether the findings show continuous GABA synthesis and degradation throughout the culture period in the cerebellar neurons. At 10 mM
AOAA, GABA synthesis from [U-13C]glutamine was not affected, indicating that transaminases are not involved in GABA synthesis and thus
excluding the putrescine pathway. At a concentration of 5 mM AOAA GABA labeling was, however, abolished, showing that glutamate
decarboxylase, which is inhibited at this level of AOAA, is responsible for GABA synthesis in the cerebellar cultures. In conclusion, the present
study shows that GABA synthesis is taking place via GAD in a subpopulation of the cerebellar neurons, throughout the culture period.
0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2006.01.005
et al., 2004). Characteristics of GABAergic neurons are GABA
transporters in vesicles and plasma membranes and glutamate
decarboxylase (GAD), the main GABA synthesizing enzyme
(Saito et al., 1974; Borden, 1996; Chaudhry et al., 1998).
GABA may also be formed via the putrescine pathway;
however, this pathway appears to be operational mostly during
early development (Seiler, 1980). Degradation of GABA takes
place via GABA-transaminase (GABA-T) and succinate
semialdehyde dehydrogenase, which are not only character-
istics of GABAergic neurons, but are ubiquitously present in
neurons and astrocytes throughout the brain (McGeer et al.,
1983).
U. Sonnewald et al. / Neurochemistry International 48 (2006) 572–578 573
Dissociated cultures of cerebella consist of a majority of
neurons with glutamatergic and some with GABAergic
phenotype since both glutamate and GABA are released in a
Ca2+ dependent manner upon depolarization of the cell
membranes (Pearce et al., 1981; Drejer et al., 1982, 1987;
Drejer and Schousboe, 1989). However, the neuronal character-
istics of cerebellar cultures can be influenced by exposure of the
cells to kainic acid (KA). This is reflected by the finding that
vesicular release of GABA can be essentially eliminated by
culturing the cells in the presence of 50 mM KA (Drejer and
Schousboe, 1989; Damgaard et al., 1996). In spite of this it has
been demonstrated that the ability of these cultures to synthesize
GABA is not affected by KA (Sonnewald et al., 2004).
Immunohistochemistry using a GAD67 antibody demonstrated
GAD-like immunostaining in �6% of the cell bodies and
additionally punctate fluorescence was observed in the processes
throughout the cultures grown in the absence of KA. Exposure of
the cerebellar neurons to KA (50 mM) eliminated the punctate
staining but had no effect on the GAD-like immunostaining in
cell bodies. Replacing KAwith a glutamate transport inhibitor to
induce excitotoxicity led to reduced levels of GAD and the
vesicular GABA transporter (Kovacs et al., 2003). Surprisingly,
the cellular content of GABA in 7-day-old cerebellar cultures
was similar to that observed in cultures of cortical neurons of the
same age which contain a dense layer of GAD positive neurons
(Sonnewald et al., 2004).
It is intriguing that GABA synthesis in these cerebellar
cultures after a 7-day culturing period reached a magnitude
comparable to that observed in neocortical neurons, since the
intensity of GAD67 immunofluorescence was considerably
lower in the cerebellar neurons. Little is known about the time
course of GABA synthesis in the latter cultures. Results
presented in this study describe the metabolism of GABA and
glutamate in cerebellar cultures. It has been shown that, during
the course of a week, both cerebellar and cortical neurons in
culture develop an enzymatic machinery analogous to that of
the brain in vivo (Drejer et al., 1985; Larsson et al., 1985).
Hence, most experiments were done on days 3, 5 and 7 in
culture. Cerebellar neurons were cultured in the presence of
50 mM KA and [1-13C]glucose or [U-13C]glutamine to monitor
GABA synthesis. Glutamine may serve as precursor for GABA
involving either GAD or the putrescine pathway. To probe the
involvement of GABA degrading enzymes in the maintenance
of GABA homeostasis, experiments were performed using
10 mM aminooxyacetic acid (AOAA) which inhibits GABA-T,
other transaminases and other pyridoxal phosphate dependent
enzymes or 100 mM g-vinyl GABA (GVG) to selectively
inhibit GABA-T (Schousboe et al., 1974; Lippert et al., 1977).
In order to inhibit GAD completely, a concentration of 5.0 mM
AOAA was used (Wu and Roberts, 1974).
2. Materials and methods
2.1. Materials
Plastic tissue culture dishes were purchased from Nunc A/S (Roskilde,
Denmark), fetal calf serum from Seralab Ltd. (Sussex, UK) and culture medium
from GIBCO BRL, Life Technologies A/S (Roskilde, Denmark). 7-day-old
mice (NMRI) were purchased from Møllegaard Breeding Center (Ejby, Den-
mark) or were obtained from the animal facility at the Department of Pharma-
cology, The Danish University of Pharmaceutical Sciences. [1-13C]Glucose and
[U-13C]glutamine (98%+ enriched) were from Cambridge Isotopes Labora-
tories (Woburn, MA, USA). KA was from Sigma Chemical Co. (St. Louis, MO,
USA) or Tocris Cookson Inc. (Ellisville, MO, USA). All other chemicals were
of the purest grade available from regular commercial sources.
2.2. Cerebellar neurons and culture conditions
All animal procedures were conducted according to national regulations.
Cerebellar cells were isolated from cerebella of 7-day-old mice (Schousboe
et al., 1989). The brain tissue was trypsinized followed by trituration in a DNase
solution containing a trypsin inhibitor from soybeans. Cells were suspended
(3 � 106 cells/ml) in a slightly modified Dulbecco’s minimum essential med-
ium (DMEM) containing 28 mM glucose and 0.45 mM glutamine, 50 mM KA
and 10% (v/v) fetal calf serum and seeded in poly-D-lysine coated culture dishes
or flasks. Six-well plates were used for HPLC and 25 cm2 flasks for liquid
chromatography mass spectrometry (LC–MS) analyses. In some cultures,
[1-13C]glucose (28 mM) or [U-13C]glutamine (0.45 mM) were used instead
of unlabeled substrate. Some cultures were exposed to 10 mM or 0.5 mM
AOAA or 100 mM GVG from day 0. Cytosine arabinoside (20 mM) was added
after 24–48 h to prevent astrocyte proliferation (Schousboe et al., 1989). After
the indicated number of days in vitro (DIV), medium was removed and cells
were washed with 0.9% saline and extracted with 70% (v/v) ethanol, followed
by centrifugation at 3000 � g for 5 min. The supernatants were lyophilized and
stored at �20 8C. In some cases media were collected, deproteinized with
ethanol (70% (v/v) final concentration), lyophilized and stored at �20 8C.
Cellular protein in the ethanol pellets was determined after re-dissolving in 1 M
KOH at 37 8C for 30 min, using the Pierce BCA protein assay with bovine
serum albumin as standard.
2.3. Acute exposure to AOAA
In order to investigate the ability of AOAA to inhibit GAD completely,
cultures were treated with 5.0 mM AOAA which was added to the medium on
day 7 in vitro. After 30 min, half (2.5 ml) of the culture medium was removed
and 1 ml fresh serum-free DMEM was added. This medium contained pyruvate
(5.0 mM, final concentration), [U-13C]glutamine (0.5 mM, final concentration)
and AOAA (5.0 mM, final concentration). After 3 h, the cultures received 8 ml
of [U-13C]glutamine (219 mM) to the incubation media (3.5 ml) to preserve an
adequate amount of the labeled precursor in the medium and the incubation was
continued for another 3 h. Control cultures were treated identically except that
AOAA was not added. At the end of the incubation period, the medium was
removed and the cells were washed twice with ice-cold PBS prior to extraction
using 70% (v/v) ethanol. The procedures for centrifugation and protein analysis
were as described above. The cell extracts were analyzed for percent labeling
employing LC–MS.
2.4. HPLC and LC–MS analyses
Glutamate and GABA were quantified in cell extracts and in some cases
GABA was quantified in the culture media by high performance liquid
chromatography (HPLC) analysis using fluorescence detection, after pre-col-
umn derivatization with o-phthaldialdehyde (Geddes and Wood, 1984). All LC–
MS analyses were performed using a Shimadzu LCMS-2010 mass spectrometer
coupled to a Shimadzu 10AVP HPLC system. The Phenomenex EZ:faast amino
acid analysis kit for LC–MS was used for analysis of labeling in glutamate and
GABA.
2.5. Data analysis
Percent 13C (atom percent excess) was determined for M + 1 (the mass of
the parent ion (M) plus 1 unit of molecular weight (Dalton) corresponding to 1
atom of 13C), M + 2, M + 3, M + 4 plus in the case of glutamate M + 5 after
correction for natural abundant 13C as described by Biemann (1962). To obtain a
U. Sonnewald et al. / Neurochemistry International 48 (2006) 572–578574
Fig. 2. The amount (nmol/well) of glutamate (A) and GABA (B) in cell extracts
of cerebellar neurons. The cells were seeded in 6-well plates and maintained in
medium containing KA (50 mM) in the presence or absence of AOAA (10 mM)
for 13 days as described in Section 2. Results are means � S.D. of 3–6 samples.
Filled squares represent the control cells, and open squares the AOAA treated
cultures. Statistically significant differences between the different days in vitro
(controls) were analyzed using ANOVA followed by the LSD (least significant
difference) post hoc test, and p < 0.05 was considered significant. For the
development in culture of glutamate contents values for DIV 7–13 were
significantly reduced compared to DIV 1–6, and for GABA DIV 3–6 were
significantly higher than DIV 1 and DIV 7–13 significantly lower than DIV 6.
No difference for GABAwas found between DIV 5 and 6. Likewise, differences
between control cultures and AOAA treated cells were assessed using the same
procedure and statistically significant differences ( p < 0.05) are indicated by
asterisks.
measure of total incorporation of 13C label, the average percent of labeled
carbon atoms for each metabolite was calculated, i.e. percent molecular carbon
labeling (MCL, for further details see Bak et al. (2006)). The [U-13C] labeling
(percent of total amount) of GABA and glutamate is the atom percent excess for
M + 4 and M + 5, respectively, following subtraction of natural abundances. All
results are presented as means � S.D. Differences between cultures of different
developmental stages or exposed to various pharmacological agents were
analyzed statistically with one-way ANOVA followed by the LSD (least
significant difference) post hoc test for multiple comparison or Student’s t-test
for two groups, and p < 0.05 was considered statistically significant.
3. Results
As a measure of cell growth the amount of protein in the
cultures was shown to almost double over a period of 4 days in
vitro and this level was maintained until day 13 (Fig. 1).
Treatment of the cultures with AOAA (10 mM) had no effect on
the protein content (Fig. 1).
The developmental patterns of the amounts (nmol/well) of
glutamate and GABA in the cerebellar neurons maintained in
the presence or absence of 10 mM AOAA during the culture
period (13 days) are shown in Fig. 2A and B. The amount of
glutamate was constant until day 6 in culture after which it was
decreasing with time until day 10 in culture. In the presence of
AOAA the amount of glutamate was lower than that of age
matched untreated cultures from days 4 to 9. The amount of
GABA more than doubled till day 5 and decreased
subsequently to a level similar to that at the beginning of the
culture period. In the presence of AOAA the GABA levels were
significantly increased compared to the age matched untreated
cultures from day 4 and throughout the culture period.
Fig. 3 shows the amount of protein (mg/well, A) and the
GABA (B) and glutamate (C) contents (nmol/mg protein) in
cerebellar neurons cultured for 3, 5 and 7 days. Culturing the cells
in medium containing GVG (100 mM) had no effect on the
protein content, whereas that of glutamate was slightly higher on
day 5 and lower on day 7 compared to the age matched untreated
cultures. The GABA content was increased in the presence of
GVG at days 5 and 7 in culture compared to age matched
untreated cultures. Culturing in the presence of 0.5 mM AOAA
Fig. 1. The amount of protein (mg/well) in cerebellar neurons. The cells were
seeded in 6-well plates and maintained in medium containing KA (50 mM) in
the presence or absence of AOAA (10 mM) for 13 days as described in Section
2. Results are means � S.D. of 3–6 samples. Filled squares represent the control
cells, and open squares the AOAA treated cultures. Differences between days 1
or 2 and subsequent days in vitro were analyzed using ANOVA followed by the
LSD (least significant difference) post hoc test, and p < 0.05 was considered
statistically significant. Protein at DIV 3–13 was statistically significantly
different from DIV 1 and 2 regardless of the presence of AOAA.
led to decreased amounts of protein and glutamate at all days
investigated and the GABA content was decreased on days 3 and
5 compared to age matched untreated cells.
Fig. 4 shows the GABA concentration (nmol/ml) in media
from neurons cultured in the absence or presence of 10 mM
AOAA for 1, 4 and 6 days. For comparison the GABA
concentration in medium (without cells) kept in the incubator
for 3 and 6 days was determined and the average value is shown
in Fig. 4. It is necessary to determine the GABA concentration
in medium without cells since fetal calf serum contains GABA
and also to evaluate the effect of incubation at 37 8C for several
days. The concentration of GABA in the medium of cells
maintained for 1 and 4 DIV was the same as that in medium
without cells. At 6 DIV the GABA concentration in the medium
was higher than that at 1 and 4 DIV. In the presence of AOAA
the GABA concentration was increased already on day 4
compared to medium from AOAA treated cultures on day 1 and
it was increased further on day 6 reaching a level significantly
higher than that of the age matched untreated cultures.
The MCL (see Section 2) of GABA and glutamate in
cerebellar neurons cultured in media containing either
[1-13C]glucose or [U-13C]glutamine is presented in Table 1.
U. Sonnewald et al. / Neurochemistry International 48 (2006) 572–578 575
Fig. 3. The amount (mg/well) of protein (A), and contents (nmol/mg protein) of
GABA (B) and glutamate (C) in cerebellar neurons cultured in 6-well plates.
Cells were cultured in medium containing KA (50 mM) in the presence or
absence of GVG (100 mM) or AOAA (0.5 mM) as described in Section 2.
Results are means � S.D. of 3–8 samples. Filled bars represent control cells.
Open and hatched bars represent GVG and AOAA treated cultures, respectively.
Statistically significant differences between treated and untreated (control)
cultures as well as between cells cultured for different number of days were
analyzed using ANOVA followed by the LSD (least significant difference) post
hoc test, and p < 0.05 was considered significant. (a) Significantly different
from the untreated cells on the same day, (*) significantly different from the
corresponding cultures on day 3, and (#) significantly different from the
corresponding cultures on day 5.
Fig. 4. The concentration (nmol/ml) of GABA in medium from cerebellar
neurons cultured in 6-well plates and maintained in medium (2 ml) containing
KA (50 mM) in the presence or absence of AOAA (10 mM). Results are corrected
for evaporation and are expressed as means � S.D. of 3–5 samples. Filled bars
represent untreated cultures (control) and open bars AOAA treated cultures. The
hatched bar represents the average of the GABA concentration in media kept in the
incubator for 3 or 6 days. It was possible to use the average since no difference was
found comparing media kept for 3 and 6 days. Statistically significant differences
between AOAA treated and not-treated (control) cultures as well as between cells
cultured for different number of days were analyzed using ANOVA followed by
the LSD (least significant difference) post hoc test, and p < 0.05 was considered
significant. (*) Significantly different from the corresponding cultures at 1 DIV,
(#) significantly different from the corresponding cultures at 4 DIV, (¤) signifi-
cantly different from medium without cells, and (a) significantly different from
control cultures at the same DIV.
Table 1
The MCL (%) of GABA and glutamate from [1-13C]glucose and [U-13C]glu-
tamine in cerebellar neurons cultured for 3 and 7 DIV
Amino acid MCL (%)
[1-13C]Glucose [U-13C]Glutamine
GABA
3 Days
AOAA 19.7 � 0.2a 12.1 � 0.4
Control 23.6 � 0.8 10.9 � 1.3
7 Days
AOAA 26.1 � 0.3a,b 4.2 � 0.2a,b
Control 28.8 � 0.2b 1.3 � 0.3b
Glutamate
3 Days
AOAA 23.2 � 0.1c 7.0 � 1.3c
Control 23.1 � 0.8 5.7 � 1.8c
7 Days
AOAA 28.3 � 0.4b,c 0.8 � 0.3b,c
Control 27.9 � 0.1b,c 1.1 � 0.5b
Cerebellar neurons were cultured in 25 cm2 flasks in media containing KA
(50 mM) in the presence or absence of AOAA (10 mM) using [1-13C]glucose or
[U-13C]glutamine as described in Section 2. The average labeling of molecular
carbon (MCL) in percent (see Section 2) of GABA and glutamate is
shown � S.D. of four cultures. Statistically significant differences between
experimental conditions and culture ages were analyzed using ANOVA fol-
lowed by the LSD (least significant difference) post hoc test, and p < 0.05 was
considered statistically significant.a Significantly different from the corresponding control cultures.b Significantly different from similarly treated cultures at 3 DIV.c Significantly different from the percent 13C labeling in GABA in cultures
treated identically.
When [1-13C]glucose was present in the medium, the MCL of
GABA was increased at 7 compared to 3 DIV, regardless of the
presence of 10 mM AOAA. MCL of GABAwas lower in AOAA
treated compared to untreated cultures kept for 3 or 7 DIV. No
differences were observed in the MCL of glutamate from
[1-13C]glucose between AOAA treated and untreated cultures
maintained for 3 or 7 DIV. However, an increase in the MCL of
glutamate was observed in cerebellar neurons cultured for 7 days
compared to 3 days. Following 3 days in culture in medium
containing [1-13C]glucose and AOAA the MCL of glutamate was
higher than that of GABA whereas in the untreated cultures no
difference was observed. After 7 days in culture in medium
U. Sonnewald et al. / Neurochemistry International 48 (2006) 572–578576
Fig. 5. The [U-13C] labeling (percent of total amount) of GABA and glutamate
in cerebellar neurons (7 DIV) incubated in medium containing [U-13C]gluta-
mine in the presence or absence of AOAA (5.0 mM) as detailed in Section 2.
Results are means � S.D. of 4 to 5 samples. Filled bars represent the untreated
and open bars the AOAA treated cells. Statistically significant differences
between these were analyzed using the unpaired two tailed Student’s t-test, and
p < 0.05 was considered statistically significant. (a) Significantly different from
the corresponding AOAA treated cultures, (b) significantly different from
[U-13C]GABA in cultures maintained under similar conditions, and n.d. means
not detectable.
containing [1-13C]glucose the MCL of glutamate was slightly
higher in AOAA treated than that observed for GABA and the
opposite relationship was observed in untreated cultures. When
[U-13C]glutamine was present in the medium from the beginning
of the culture period, the MCL of both GABA and glutamate was
decreased in cells cultured for 7 compared to 3 DIV. The MCL of
GABA was higher than that of glutamate regardless of the
experimental conditions, except at day 7 in cultures not exposed
to AOAA. Furthermore, the MCL of GABAwas increased in the
AOAA treated cells in 7-day-old cultures but unchanged in 3-
day-old cultures. This is in contrast to cells cultured in the
presence of [1-13C]glucose, in which label was decreased in the
presence of AOAA both at 3 and 7 DIV. However, as shown in
Fig. 5, when AOAA (5 mM) was added to the medium on day 7,
conversion of [U-13C]glutamine to [U-13C]glutamate took place
whereas no [U-13C]GABA was detected. In these cultures
pyruvate was added to the culture medium together with
[U-13C]glutamine to ensure TCA cycle metabolism and cell
survival in the presence of AOAA which prevents a continuous
oxidation of glucose due to the inhibition of the malate aspartate
shuttle (McKenna et al., 2006). Cell viability was checked by
microscopic inspection of the cultures and cell morphology was
not affected by the AOAA treatment (results not shown).
4. Discussion
The present study demonstrates GABA synthesis via GAD
in cerebellar neurons throughout the first week in culture. The
cellular content of GABA in the cerebellar neurons more than
doubled during the first 5 days in culture, whereas that of
glutamate remained unchanged. This was accompanied by an
increase in the amount of protein. It should be noted that the
number of neurons does not increase after the brain tissue is
removed from the animals and subsequently seeded in the
culture dishes. Actually, approximately 50% of the seeded cells
die (Westergaard et al., 1991). Thus, the increase in protein
reflects cell growth and possibly differentiation. The enzymatic
machinery of cerebellar neurons after 1 week in culture is
comparable to that observed in the brain in vivo (Drejer et al.,
1985). After day 5 in culture the amount of GABA and
glutamate inside the cells decreased slightly until the end of the
culture period (day 13). However, GABA content in the
medium increased with time. This indicates that GABA is
released from the cerebellar cells in a non-depolarization
dependent manner, presumably via reversal of transporters and
may reflect its functional importance during differentiation
(Belhage et al., 1985; Waagepetersen et al., 1999). Further-
more, GABA release is a prerequisite for its neurotrophic action
and may also play a role in neuroprotection.
Synthesis of GABA during the first 3 days was demonstrated
by the finding that incubation with either [1-13C]glucose or
[U-13C]glutamine led to formation of labeled GABA. It may be
conceivable that GABA synthesis could be especially prominent
during the first few days in culture since it has been reported that
in hippocampus, GAD67 is expressed in the mossy fibers of the
developing rat brain, whereas in adults, GAD67 was no longer
detectable, unless seizures were induced (Maqueda et al., 2003).
To investigate the possibility that GABA synthesis is not only
taking place during the early phase of the culturing period but
continues also during later stages, [U-13C]glutamine was added
on day 7 in culture and both GABA and glutamate labeling was
pronounced. Furthermore, when [1-13C]glucose was present in
the culture medium for 3 and 7 days, labeling on day 7 was clearly
higher than on day 3. Labeling from [U-13C]glutamine under the
same conditions was less pronounced than that from glucose,
which may reflect that the amount of [U-13C]glutamine available
in the medium was insufficient to sustain neuronal metabolism
for 7 days. That this may be the case is supported by the finding
that when [U-13C]glutamine was added on day 7, GABA labeling
was much higher.
To investigate the enzymatic pathways responsible for
GABA synthesis and degradation, enzyme inhibitors were
added to the culture medium. The enzyme responsible for
degradation of GABA, GABA-T, is inhibited by GVG (Lippert
et al., 1977). As expected (Gram et al., 1988), GVG at a
concentration of 100 mM increased the GABA content of the
cultures. GVG had no effect on the protein content which may
indicate that the GABA concentration even in the absence of
GVG was adequate for maintenance of normal neuronal
growth. This is supported by the finding that GABA at 50 mM, a
value comparable to that observed in the present study, acts as a
trophic factor in the development of cerebellar neuronal
cultures (Hansen et al., 1984). The transaminase inhibitor
AOAA was used at 10 mM, a concentration sufficient to inhibit
mainly GABA-T (Schousboe et al., 1974) but also other
transaminases (Kihara and Kubo, 1989). As expected, the
amount of GABA increased with time even more than in the
absence of AOAA whereas the glutamate concentration
decreased compared to that in untreated cells. The latter
finding may be compatible with the previous demonstration that
U. Sonnewald et al. / Neurochemistry International 48 (2006) 572–578 577
biosynthesis of neurotransmitter glutamate in the glutamatergic
neurons in these cultures is dependent upon the function of the
malate aspartate shuttle which involves transamination
(Palaiologos et al., 1988). Glutamate formed via transamination
seems to be important for GABA labeling from [1-13C]glucose
(for pathway see Brenner et al. (2005)) since such labeling was
decreased in the presence of 10 mM of AOAA. Conversion of
a-ketoglutarate to glutamate is mostly achieved by transamina-
tion, which will be blocked by AOAA. However, glutamate
dehydrogenase is also present in the cerebellar neurons
(Zaganas et al., 2001) and can convert a-ketoglutarate to
glutamate. The efficiency of this process is evident from the
unchanged glutamate labeling in the presence of 10 mM
AOAA. This, together with a decreased GABA labeling, points
towards compartmentation of glutamate metabolism, indicating
GABA synthesis in a different cellular compartment from
where the majority of glutamate synthesis is taking place.
There are two known pathways for conversion of glutamine
to GABA. One is called the putrescine pathway (Seiler, 1980)
which involves transamination and the other conversion of
glutamine to glutamate and subsequent decarboxylation to
GABA which is referred to as the GAD pathway. Labeling of
GABA from [U-13C]glutamine was not affected by 10 mM
AOAA, indicating that transamination is not involved in the
process. Thus, it appears likely that the GAD pathway is
responsible for GABA synthesis from glutamine in the
cerebellar cultures. This, together with the fact that metabolism
of glutamate and GABA seems compartmentalized as
mentioned above, indicates that GABA synthesis takes place
in the �6% GAD positive cells observed by Sonnewald et al.
(2004).
An increase in the AOAA concentration from 10 mM to
0.5 mM led to a pronounced decrease in the GABA and
glutamate content as well as the amount of protein at 3 and 5
DIV. As mentioned above, in the presence of the transaminase
blocker, the decrease in glutamate content was expected. The
large decrease in GABA may to a certain extent be explained by
partial inhibition of GAD in the presence of 0.5 mM AOAA
(Wu and Roberts, 1974) and the decrease in protein content
may to some extent reflect the decrease in the availability of the
neurotrophic agent GABA. However, it is conceivable that an
impaired oxidative metabolism of glucose played a prominent
role in the large decrease of the protein content since this
metabolism is dependent upon the malate aspartate shuttle
which was inhibited by 0.5 mM AOAA (Kauppinen et al.,
1987). An impaired glucose metabolism will affect neurons and
therefore it may indirectly contribute to the decrease in
glutamate as well as GABA contents. Interestingly, on day 7 in
culture, the GABA content was not different from that of age
matched untreated cells, whereas glutamate and the protein
content were lower.
To obtain further information about GABA synthesis,
AOAA was used at a concentration of 5 mM, which is expected
to block both GAD and transaminases completely (Wu and
Roberts, 1974; Kihara and Kubo, 1989). Indeed, GABA
labeling from [U-13C]glutamine was totally abolished even
though glutamate was extensively labeled.
In conclusion, the present results show that GABA synthesis
is taking place via GAD in a subpopulation of the cerebellar
neurons, throughout the culture period. Labeling of GABA
occurs from both [1-13C]glucose and [U-13C]glutamine and can
be blocked by AOAA. Moreover, it is confirmed that net
synthesis of glutamate is dependent on the activity of the malate
aspartate shuttle.
Acknowledgements
The expert technical assistance by Mrs. Kirsten Thuesen and
Bente Urfjell is cordially acknowledged. The Danish State
Medical Research Council (22-03-0250; 22-04-0314), the
Norwegian Epilepsy, Hørslev, Lundbeck and Novo Nordisk
Foundations are thanked for financial support.
References
Bak, L.K., Schousboe, A., Sonnewald, U., Waagepetersen, H.S., 2006. Glucose
is necessary to maintain neurotransmitter homeostasis during synaptic
activity in cultured glutamatergic neurons. J. Cereb. Blood Flow Met.
[Epub ahead of print].
Belhage, B., Hansen, G.H., Elster, L., Schousboe, A., 1985. Effects of gamma-
aminobutyric acid (GABA) on synaptogenesis and synaptic function.
Perspect. Dev. Neurobiol. 5, 235–246.
Biemann, K., 1962. Mass spectrometry. In: Organic Chemistry Applications,
McGraw, New York, pp. 223–227.
Brenner, E., Kondziella, D., Haberg, A., Sonnewald, U., 2005. Impaired
glutamine metabolism in NMDA receptor hypofunction induced by
MK801. J. Neurochem. 94, 1594–1603.
Borden, L.A., 1996. GABA transporter heterogeneity: pharmacology and
acid, a selective catalytic inhibitor of 4-aminobutyric-acid aminotransferase
in mammalian brain. Eur. J. Biochem. 74, 441–445.
Maqueda, J., Ramirez, M., Lamas, M., Gutierrez, R., 2003. Glutamic acid
decarboxylase (GAD)67, but not GAD65, is constitutively expressed during
development and transiently overexpressed by activity in the granule cells of
the rat. Neurosci. Lett. 353, 69–71.
McGeer, P.L., McGeer, E.G., Nagai, T., 1983. GABAergic and cholinergic
indices in various regions of rat brain after intracerebral injections of folic
acid. Brain Res. 260, 107–116.
McKenna, M., Waagepetersen, H.S., Schousboe, A., Sonnewald, U., 2006.
Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial
transfer of reducing equivalents: current evidence and pharmacological
tools. Biochem. Pharmacol. 71, 399–407.
Palaiologos, G., Hertz, L., Schousboe, A., 1988. Evidence that aspartate
aminotransferase activity and ketodicarboxylate carrier function are
essential for biosynthesis of transmitter glutamate. J. Neurochem. 51,
317–320.
Pearce, B.R., Currie, D.N., Beale, R., Dutton, G.R., 1981. Potassium-stimu-
lated, calcium-dependent release of [3H]GABA from neuron- and glia-
enriched cultures of cells dissociated from rat cerebellum. Brain Res. 206,
485–489.
Saito, K., Barber, R., Wu, J., Matsuda, T., Roberts, E., Vaughn, J.E., 1974.
Immunohistochemical localization of glutamate decarboxylase in rat cer-
ebellum. Proc. Natl. Acad. Sci. U.S.A. 71, 269–273.
Schousboe, A., Wu, J.Y., Roberts, E., 1974. Subunit structure and kinetic
properties of 4-aminobutyrate-2-ketoglutarate transaminase purified from
mouse brain. J. Neurochem. 23, 1189–1195.
Schousboe, A., Meier, E., Drejer, J., Hertz, L., 1989. Preparation of primary
cultures of mouse (rat) cerebellar granule cells. In: Shahar, A., de Vellis,
J., Vernadakis, A., Haber, B. (Eds.), A Dissection and Tissue Culture
Manual for the Nervous System. Alan R. Liss, New York, pp. 183–186.
Seiler, N., 1980. On the role of GABA in vertebrate polyamine metabolism.
Physiol. Chem. Phys. 12, 411–429.
Sonnewald, U., Olstad, E., Qu, H., Babot, Z., Cristofol, R., Sunol, C., Schous-
boe, A., Waagepetersen, H., 2004. First direct demonstration of extensive
GABA synthesis in mouse cerebellar neuronal cultures. J. Neurochem. 91,
796–803.
Waagepetersen, H.S., Sonnewald, U., Schousboe, A., 1999. The GABA para-
dox: multiple roles as metabolite, neurotransmitter, and neurodifferentiative
agent. J. Neurochem. 73, 1335–1342.
Westergaard, N., Fosmark, H., Schousboe, A., 1991. Metabolism and release of
glutamate in cerebellar granule cells cocultured with astrocytes from
cerebellum or cerebral cortex. J. Neurochem. 56, 59–66.
Wu, J.Y., Roberts, E., 1974. Properties of brain L-glutamate decarboxylase:
inhibition studies. J. Neurochem. 23, 759–767.
Zaganas, I., Waagepetersen, H.S., Georgopoulos, P., Sonnewald, U., Plaitakis,
A., Schousboe, A., 2001. Differential expression of glutamate dehydrogen-
ase in cultured neurons and astrocytes from mouse cerebellum and cerebral
cortex. J. Neurosci. Res. 66, 909–913.
Paper 3
Glutamate is preferred over glutamine for intermediary
metabolism in cultured cerebellar neurons
Olstad E, Qu H and Sonnewald U.
J Cereb Blood Flow Metab (2006) in press
Glutamate is preferred over glutamine forintermediary metabolism in cultured cerebellarneurons
Elisabeth Olstad1, Hong Qu2 and Ursula Sonnewald1
1Department of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway;2Department of Anatomy, Centre for Molecular Biology and Neuroscience, University of Oslo, Oslo, Norway
The glutamate–glutamine cycle is thought to be of paramount importance in the mature brain;however, its significance is likely to vary with regional differences in distance between astrocyte andsynapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellarneurons, mainly consisting of glutamatergic granule cells. Cells were incubated in mediumcontaining [U-13C]glutamate or [U-13C]glutamine in the presence and absence of unlabeledglutamine and glutamate, respectively. Cell extracts and media were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography combined with massspectrometry (GC/MS). Both [U-13C]glutamate and [U-13C]glutamine were shown to be excellentprecursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycleintermediates. Labeling from [U-13C]glutamate was higher than that from [U-13C]glutamine in allmetabolites measured. The presence of [U-13C]glutamate plus unlabeled glutamine in theexperimental medium led to labeling very similar to that from [U-13C]glutamate alone. However,incubation in medium containing [U-13C]glutamine in the presence of unlabeled glutamate almostabolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substratefor intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activityshowed more prominent cycling from [U-13C]glutamine than from [U-13C]glutamate. Labeling ofsuccinate was lower than that of the other TCA cycle intermediates, indicating an active role of thec-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons relymore on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 October 2006; doi:10.1038/sj.jcbfm.9600400
Glutamate is a multipurpose amino acid in themature central nervous system. It is not only themajor excitatory neurotransmitter, in addition ittakes part in transamination and thus nitrogenhomeostasis and is the precursor for other importantmolecules, including the main inhibitory neuro-transmitter, g-amino butyric acid (GABA). Althoughglutamate is ubiquitous in all parts of the centralnervous system and present in large amounts in thebrain, it is of critical importance that brain gluta-
mate homeostasis is strictly controlled. The extra-cellular concentration of glutamate needs to be keptlow, both to increase the signal-to-noise ratio inbinding of transmitter substance in the synaptic cleftand to prevent excitotoxicity caused by excessiveexcitation of glutamate receptors and subsequentcell injury or death (for references, see Daikhin andYudkoff, 2000). Rapid transport of glutamate fromthe synaptic cleft is performed through several typesof specific transporter proteins, and uptake intoastrocytes surrounding the synapse is believed to bemore important than reuptake into the presynapticneuron (Schousboe et al, 1977; Danbolt, 2001). Thusneurons experience a net loss of glutamate, whichmust be replenished by astrocytes because of thelack of the anaplerotic enzyme pyruvate carboxylasein neurons (Shank et al, 1985). This constitutes thebasis for the pathway known as the glutamate–glutamine cycle (Berl and Clarke, 1983) in which
Received 19 June 2006; revised 10 August 2006; accepted 11August 2006
Correspondence: Professor U Sonnewald, Department of Neuro-science, Faculty of Medicine, Olav Kyrres gate 3, N-7489Trondheim, Norway.E-mail: [email protected]
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10& 2006 ISCBFM All rights reserved 0271-678X/06 $30.00
www.jcbfm.com
neurotransmitter released from neurons is taken upby surrounding astrocytes, where it is converted toglutamine by the glial-specific enzyme glutaminesynthetase (Norenberg and Martinez-Hernandez,1979). Glutamine is not neuroactive and can movein the extracellular space without interfering withreceptors, and is the most abundant amino acid inblood and cerebral spinal fluid with a concentrationof B0.5 mmol/L (Grill et al, 1992; White et al, 2004).Glutamine uptake into neurons is mediated bydifferent general amino-acid transporters (Su et al,1997; Dolinska et al, 2004). In neurons, glutaminecan be converted into glutamate by the enzymephosphate activated glutaminase and act as pre-cursor for restoring the neurotransmitter pool,completing the glutamate–glutamine cycle. Indeed,several studies have confirmed the importance ofglutamine as precursor for neurotransmitter gluta-mate (for a review, see Peng et al, 1993). Asdescribed, the cycle necessitates extensive inter-action between neurons and astrocytes. This will,however, vary because of differences in the numberof glial cells per neuron and also in respect tohow closely the astrocytes envelop the synapses.Thus, it can be expected that the importance of theglutamate–glutamine cycle varies with location inthe brain.
The present study is aimed at evaluating the roleof the glutamate–glutamine cycle in cultured cere-bellar neurons. It has been shown that after 7 days invitro, these mainly glutamatergic cultures expressglutamate and glutamine metabolizing enzymesanalogous to the brain in vivo (Drejer et al, 1985).The cells were incubated in medium containing[U-13C]glutamate or [U-13C]glutamine in the pre-sence and absence of unlabeled glutamine andglutamate, respectively. High-performance liquidchromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis of cell extractsand media revealed that both [U-13C]glutamate and[U-13C]glutamine were excellent precursors forsynthesis of neuroactive amino acids and tricar-boxylic acid (TCA) cycle intermediates in thesecultures. However, glutamate was shown to be thepreferred substrate.
Materials and methods
Materials
NMRI mice were obtained from Taconic M&B (Copenha-gen, Denmark). Plastic tissue culture dishes were pur-chased from Nunc A/S (Roskilde, Denmark) and fetal calfserum from Seralab Ltd. (Sussex, UK). Culture medium,glutamate receptor antagonists DNQX (6,7-dinitroquinoxa-line-2,3-dione) and D-AP5 (D-2-amino-5-phosphonopenta-noic acid) were from Sigma Chemical Co. (St Louis, MO,USA). [U-13C]Glutamate and [U-13C]glutamine werefrom Cambridge Isotope Laboratories (Woburn, MA,USA), and the GC/MS derivatization reagent MTBSTFA(N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide)+ 1%
t-BDMS-Cl (tert-butyldimethylchlorosilane) was purchasedfrom Regis Technologies, Inc. (Morton Grove, IL, USA).All other chemicals were of the purest grade availablefrom regular commercial sources.
Cell Cultures
Cerebellar neurons were isolated and cultured from 7-day-old mice as described by Schousboe et al (1989). Briefly,tissue was trypsinized followed by trituration in a DNasesolution containing a trypsin inhibitor from soybeans.Cells were suspended (2.5� 106 cells/ml) in a modifiedDulbecco’s minimum essential medium containing24.5 mmol/L KCl, 31 mmol/L glucose, 7 mmol/L p-amino-benzoic acid, 0.05 mmol/L kainic acid, and 10% (v/v) fetalcalf serum, and seeded in poly-D-lysine coated Petridishes (2 ml/35 mm). After 48 h in culture, 20 mmol/L(final concentration) cytosine arabinoside was added tothe medium to prevent astrocytic proliferation.
Experiments Using [U-13C]Glutamate and[U-13C]Glutamine for Gas Chromatography/MassSpectrometry Analysis
The culture medium was removed from 7-day-old culturesand the cells were incubated for 2 h at 371C in 2 ml serum-free experimental medium (prepared without glutamine)containing 3 mmol/L glucose and either no additions (forHPLC analysis), [U-13C]glutamate (0.25 mmol/L), [U-13C]glutamine (0.50 mmol/L), [U-13C]glutamate (0.25 mmol/L)plus unlabelled glutamine (0.50 mmol/L) or [U-13C]glutamine (0.50 mmol/L) plus unlabelled glutamate(0.25 mmol/L). To avoid toxic effects of glutamate duringthe incubation period, two glutamate receptor antagonistsDNQX (25 mmol/L), a selective antagonist at the a-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) andkainate receptor subtypes, and D-AP5 (100 mmol/L), an N-methyl-D-aspartate (NMDA) antagonist, were also presentin the experimental medium (Frandsen et al, 1989). After2 h, the experimental medium was collected and the cellswere washed twice with cold phosphate-buffered salineand extracted with 70% (v/v) ethanol. The cell extractswere scraped off the dishes and centrifuged at 10,000g for15 min to separate the metabolites from the insolubleproteins. The supernatants (cell extracts) were dividedinto two parts, one directly analyzed with HPLC and theother lyophilized for subsequent sample preparation forGC/MS analysis. Cellular protein in the ethanol pelletswas determined after dissolving in 1 mol/L KOH at 371Cfor 60 mins, using the Pierce BCA (Pierce, Rockford, IL,USA) protein assay with bovine serum albumin asstandard.
High-Performance Liquid Chromatography
Amino acids in cell extracts and experimental mediawere quantified by HPLC on a Hewlett Packard 1100system (Agilent Technologies, Palo Alto, CA, USA).The amino acids were precolumn derivatized with
Glutamate metabolism in cerebellar neuronsE Olstad et al
2
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
o-phthaldialdehyde (Geddes and Wood, 1984) and subse-quently separated on a ZORBAX SB-C18 (4.6� 250 mm,5 mm) column from Agilent using a phosphate buffer(50 mmol/L, pH = 5.9) and a solution of methanol (98.75%)and tetrahydrofuran (1.25%) as eluents. The separatedamino acids were detected with fluorescence and com-pared with a standard curve derived from standardsolutions of amino acids run after every 12 samples.
Gas Chromatography/Mass Spectrometry
Lyophilized cell extracts were redissolved in HCL(10 mmol/L), adjusted to pH < 2 with 6 mol/L HCL, anddried under atmospheric air. The amino acids wereextracted into an organic phase of ethanol and benzeneand dried again under atmospheric air before derivatiza-tion with MTBSTFA in the presence of 1% t-BDMS-Cl(Mawhinney et al, 1986). The samples were analyzed on aHewlett Packard 5890 Series II gas chromatograph linkedto a Hewlett Packard 5972 Series mass spectrometer.
Data Analysis
Peaks from MS spectra were integrated, and atom percentexcess (13C) of glutamate, glutamine, GABA, succinate,malate, aspartate, and citrate was determined aftercalibration using unlabeled standard solutions (Biemann,1962). Results from HPLC quantification of amino acids incell extracts were combined with values of atom percentexcess obtained from GC/MS to give nmol/mg protein ofdifferent 13C labeled isotopomers of glutamate, aspartate,and GABA. Consumption of [U-13C]glutamate and[U-13C]glutamine was calculated by subtracting the re-maining amounts of the two amino acids measured in theexperimental medium from the amounts added followedby correction for the amount of cellular protein in theculture. Results are presented as means7s.d. Differencesbetween groups were analyzed statistically using one-wayanalysis of variance followed by the least significantdifference post hoc test, and P < 0.05 was consideredstatistically significant.
Results
Cerebellar neurons were incubated in medium withdifferent additions: no addition (control group),[U-13C]glutamate, [U-13C]glutamine, [U-13C]gluta-mate plus glutamine, and [U-13C]glutamine plusglutamate (see Materials and methods). Since HPLCanalysis does not distinguish between isotopomers,the latter two groups were combined in Table 1,which shows the cellular content of selected aminoacids in cerebellar neurons and the consumption ofglutamate and glutamine. Compared with control,adding glutamate to the experimental medium led toincreased intracellular levels of aspartate, gluta-mate, glutamine, and GABA. Cultures incubatedwith glutamine contained increased levels of GABAcorresponding to the increase seen when glutamatewas added. Aspartate and glutamate also increasedas a response to glutamine addition, but not to thesame extent as when glutamate was added. Asexpected, intracellular glutamine concentration alsoincreased when glutamine was added to the med-ium. When both glutamate and glutamine wereadded to the experimental medium, aspartate,glutamate, and glutamine levels were increasedmore than when the two amino acids were addedindividually, whereas GABA content was increasedto the same extent as when glutamate and glutaminewere added alone. Quantification of glutamate andglutamine in the experimental media showed thatglutamate consumption was much higher thanglutamine consumption despite the medium con-centration of glutamine being twice that of gluta-mate. Glutamate consumption was unaffected by thepresence of glutamine, whereas the consumption ofglutamine was reduced by nearly 50% whenglutamate was added to the medium.
[U-13C]Glutamate or [U-13C]glutamine from theexperimental medium enters neurons through spe-cific transporter proteins. Once inside the cells,[U-13C]glutamine can be converted to [U-13C]gluta-mate, which can be decarboxylated to uniformlylabeled GABA in GABAergic neurons, or in all cells,
Table 1 Cellular content of selected amino acids (nmol/mg protein) in extracts of cultured cerebellar neurons and consumption ofglutamate and glutamine (mmol/mg protein/2 h) from the experimental medium
Cerebellar neurons were incubated for 2 h in medium containing 3 mmol/L glucose, 25 mmol/L DNQX, and 100 mmol/L D-AP5 and either no addition (Ctr(control), n = 3), 0.25 mmol/L glutamate (Glu, n = 6), 0.50 mmol/L glutamine (Gln, n = 6), or 0.25 mmol/L glutamate plus 0.50 mmol/L glutamine (Glu+Gln,n = 12), for details see Materials and methods. Results are presented as means7s.d., and P < 0.05 was considered statistically significant.aDifferent from the Ctr group.bDifferent from the Glu group.cDifferent from the Gln group.
Glutamate metabolism in cerebellar neuronsE Olstad et al
3
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
be turned into a-[U-13C]ketoglutarate and be meta-bolized in the TCA cycle for energy production andmetabolite synthesis. The 13C label from the pre-cursor’s carbon skeleton will in the latter case bedistributed among the TCA cycle metabolites andGABA as illustrated in Figure 1.
The labeling of glutamate, GABA, succinate,malate, aspartate, and citrate as detected by GC/MSis presented in Figure 2 as atom percent excess (%labeling). When [U-13C]glutamate is turned intoa-[U-13C]ketoglutarate and enters the TCA cycle,[1,2,3-13C]glutamate (M + 3) is formed after one turn.This isotopomer results from condensation ofuniformly labeled oxaloacetate (OAA) with un-labeled acetyl-CoA forming [3,4,5,6-13C]citrate(Figure 1), which can be turned into [1,2,3,6-13C]iso-citrate, a-[1,2,3-13C]ketoglutarate and finally into[1,2,3-13C]glutamate. After another turn in the TCAcycle, half of the glutamate formed will be[1,2-13C]glutamate (M + 2) and half [3-13C]glutamate(M + 1). After a third turn, 25% of glutamate will beunlabeled, whereas 75% will be labeled either in the1, 2, or 3 position (M + 1) (Figure 1).
From [U-13C]glutamate entering the TCA cycle viaa-[U-13C]ketoglutarate, uniformly labeled succinate,malate, and OAA are formed (Figure 1), and OAAcan be transaminated into aspartate. All of thesecompounds have four C atoms, and their [U-13C]isoforms have the mass M + 4 and are presented inFigure 2. [U-13C]OAA can, as already mentioned,condense with unlabeled acetyl-CoA to form[3,4,5,6-13C]citrate with six carbon atoms, four of
which are 13C (M + 4). Hence, the mass M + 4represents the first turn in the TCA cycle for allthese compounds. In the next turn, they will allcontain two labeled C atoms and appear as M + 2,and in the third turn, they will contain one labeled Catom (M + 1) (Figure 1).
As shown in Figure 2, labeling from [U-13C]gluta-mate (column A) and [U-13C]glutamine (column B)was substantial, with glutamate giving the highestpercent labeling in all metabolites analyzed. When[U-13C]glutamate and unlabeled glutamine wereadded to the experimental medium (column C),percent label decreased only slightly compared withwhen [U-13C]glutamate was added alone (columnA). However, when [U-13C]glutamine and unlabeledglutamate were added to the medium, labeling wasalmost abolished for all metabolites (column D).Figure 2 shows that labeling of glutamate andmalate, aspartate and citrate was high ( > 65% from[U-13C]glutamate and 40% to 60% from [U-13C]glu-tamine (in the absence of glutamate)), whereas thatof succinate was much lower ( < 30% labeling fromglutamate and < 10% from glutamine). Although thepercent labeling in succinate was lower, the patternwas the same as for the other TCA metabolites.
GABA can be formed from glutamate in GABAergic neurons, which constitute about 6% of cere-bellar neuronal cultures (Sonnewald et al, 2004).GABA formed directly from [U-13C]glutamate isuniformly labeled (M + 4). From [1,2,3-13C]glutamate(after one turn in the TCA cycle), [3,4-13C]GABA isformed (M + 2). Another turn in the TCA cycle for
GLN/GLU/αKG
SUC/FUM/MAL/OAA/ASP
Acetyl CoA
CIT
αKG/GLU
GABA
6
54321
1234
12345
54321
1234
12
3rd turn2nd turn1st turn
Figure 1 Schematic representation of possible isotopomers of various metabolites arising from [U-13C]glutamate or [U-13C]glutaminevia the three first turns in the TCA cycle in neurons: K represents 13C and J represents 12C atoms; GLN: glutamine; GLU:glutamate; aKG: a-ketoglutarate; SUC: succinate; FUM: fumarate; MAL: malate; OAA: oxaloacetate; ASP: aspartate; CIT: citrate;GABA: g-amino butyric acid.
Glutamate metabolism in cerebellar neuronsE Olstad et al
4
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
the glutamate carbon skeleton and subsequentformation of GABA will result in labeling in one Catom in GABA (M + 1), from both [1,2-13C] and[3-13C]glutamate, [4-13C] and [3-13C]GABA, respec-tively. The same two isotopomers are also formedafter three turns in the TCA cycle (Figure 1). Figure 2shows that the labeling of GABA analyzed by GC/MS was lower than that of glutamate, malate,aspartate, and citrate. Total labeling from [U-13C]glu-tamate was B30% regardless of the presence ofunlabeled glutamine. Significantly less (17%)GABA was labeled from [U-13C]glutamine alone,and this was further reduced in the presence ofunlabeled glutamate (6%).
Intracellular amounts of glutamate, aspartate, andGABA as quantified by HPLC are shown in Figure 3.The amounts of glutamate and aspartate variedconsiderably with addition of glutamate or gluta-mine in the experimental medium, but followed thesame pattern, whereas GABA concentration wasindependent of glutamate and glutamine content inmedium. Results from mass spectrometry provide
information about percent labeling, which com-bined with information about amounts of metabo-lites gives quantitative data. Amounts of different13C labeled isotopomers of glutamate, aspartate, andGABA from the first two turns in the TCA cycle areshown in Figure 3. The intracellular amount ofuniformly labeled glutamate was 248 nmol/mg pro-tein after incubation with [U-13C]glutamate. Thiswas reduced to 32 and 17 nmol/mg protein whenglutamine was the labeled precursor, in the absenceand presence of unlabeled glutamate, respectively.When labeled glutamate was added in the presenceof unlabeled glutamine, the amount of intracellularuniformly labeled glutamate was, however, in-creased to 279 nmol/mg protein. The same trendwas seen in [U-13C]aspartate, although the amountswere B50% of those of [U-13C]glutamate. These twoexcitatory amino acids also showed similarities inthe next turn in the TCA cycle. GABA had a muchlower labeling from both labeled precursors thanglutamate and aspartate, labeling from [U-13C]gluta-mine being lower than that from [U-13C]glutamate.
Glutamate
0
20
40
60
80
100
A B C D
% la
bel
ing
M+5 M+3 M+2 M+1
Malate
A B C D
Aspartate
A B C D
Citrate
0
20
40
60
80
100
A B C D
% la
bel
ing
0
20
40
60
80
100%
lab
elin
g
0
20
40
60
80
100
% la
bel
ing
M+4 M+2 M+1M+4 M+2 M+1M+4 M+2 M+1
GABA
0
5
10
15
20
25
30
35
A B C D
% la
bel
ing
0
5
10
15
20
25
30
35
% la
bel
ing
M+4 M+2 M+1
Succinate
A B C D
*
a,b
a,b
b
b
a
a
a
a
a
a
a
b
b
b
a
a
b
b
a
a
a
a, b
b
b
a
a
a
a, b
a, b
b
a
a
a
b
b
*#
* * *
a,b
M+4 M+2 M+1
Figure 2 Percent labeling in glutamate, GABA, succinate, malate, aspartate and citrate as detected by GC/MS in cell extracts ofcultured cerebellar neurons after incubation with [U-13C]glutamate (column A), [U-13C]glutamine (column B), [U-13C]glutamate plusunlabeled glutamine (column C) or [U-13C]glutamine plus unlabeled glutamate (column D), for details see Materials and methods.Results are presented as means + s.d. in atom percent excess, and P < 0.05 was considered statistically significant. *All masses aredifferent from the corresponding masses in the other groups. #M + 4 is different from the groups incubated with [U-13C]glutamatewith and without glutamine, the other masses are different from all other groups. aDifferent from the corresponding mass in the Agroup; bdifferent from the corresponding mass in the B group; cdifferent from the corresponding mass in the C group.
Glutamate metabolism in cerebellar neuronsE Olstad et al
5
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
Discussion
Metabolism of glutamate and glutamine in thebrain is closely connected via the glutamate–glutamine cycle, in which neurotransmitterglutamate taken up from the synaptic cleft byastrocytes is converted to glutamine and trans-ported back to neurons as a precursor for theneurotransmitter pool (Berl and Clarke, 1983).Similar mechanisms operate in the glutamate–glutamine–GABA cycle (van den Berg and Garfinkel,1971; Sonnewald et al, 1993b). However, reuptakeinto the presynaptic neuron is believed to bethe preferred mechanism for removal of GABAfrom the synapse (Schousboe, 2003). To whichextent glutamine and thus this cycle is impor-tant for cultured cerebellar neurons has beenexplored in the present study by comparing[U-13C]glutamate and [U-13C]glutamine as substratesfor intermediary metabolism. In our model, additionof glutamine to the neuronal cultures mimicsastrocyte–neuronal interactions in the brain, withthe advantage of isolating neuronal from glialmetabolism and thus making interpretation ofresults unambiguous.
[U-13C]Glutamate and [U-13C]Glutamine Metabolism
Glutamine has been shown to be an excellentprecursor for the neurotransmitters glutamateand GABA, both in cultured cerebellar neurons(Waagepetersen et al, 2005), cortical neurons(Westergaard et al, 1995), and in freshly isolatedcortical synaptosomes from rat brain (Yudkoff et al,
1989; Sonnewald and McKenna, 2002). Labelfrom [U-13C]glutamine was also found in glutamateand GABA in the present study. Moreover, labelingof TCA cycle intermediates and aspartate wasdetected, showing that the carbon skeleton of[U-13C]glutamine entered the TCA cycle. Thepresence of isotopomers from subsequent turns alsoshowed that the carbon skeleton stayed in the TCAcycle. This confirms that the glutamate–glutaminecycle does not operate in a stochiometric fashion(McKenna et al, 1993, 1994; Sonnewald et al, 1993a)and that glutamine is readily oxidized by cerebellarcultures for energy.
Exogenous glutamate has also been used to labelneuronal metabolites. Westergaard et al (1995)showed that in cultured cortical neurons, whichare predominantly GABAergic, incubation with[U-13C]glutamate gave high enrichment in aspartatein addition to labeling of GABA. Incubating cere-bellar neurons in medium containing [U-13C]gluta-mate in the present study led not only to uniformlylabeled intracellular aspartate and glutamate, but tosome extent, the carbon skeleton also stayed in theTCA cycle for several turns. This is in accordancewith similar studies of cerebellar neurons analyzedwith magnetic resonance spectroscopy (Sonnewaldet al, 1996; Santos et al, 2006). Using the moresensitive method mass spectrometry, extensivelabeling of TCA cycle intermediates and GABAwas also detected in the present study. Labeling ofGABA in cerebellar cultures has earlier been shownby Qu et al (2000). The present study confirms that[U-13C]glutamate serves as an excellent precursor forintermediary metabolism in cultured cerebellarneurons.
GABA
ab
a,b,cab
a,b,c0
2
4
6
8
10
12
14
C D
nm
ol/m
g p
rote
in
Aspartate
a
a a
0
50
100
150
200
250
300
350
A B A BC Dn
mo
l/mg
pro
tein
Glutamate
a, ba, b
a, b
a, b
a
a a, b, ca, b, c
a, b, ca, b, c
a
0
100
200
300
400
500
600
A B C D
nm
ol/m
g p
rote
in
Total (HPLC) M+5 M+3
a, ba, b
a, b
a, b
Total (HPLC) M+4 M+2 Total (HPLC) M+4 M+2
Figure 3 The amounts of glutamate, aspartate, and GABA (white bars), the amounts of M + 5 ([U-13C]glutamate), M + 4([U-13C]aspartate, [U-13C]GABA) (black bars) and the amounts of M + 3 ([1,2,3-13C]glutamate), M + 2 ([1,2-13C] and[3,4-13C]aspartate) and M + 2 ([3,4-13C]GABA) (grey bars) in cell extracts of cultured cerebellar neurons after incubation with[U-13C]glutamate (column A), [U-13C]glutamine (column B), [U-13C]glutamate plus unlabeled glutamine (column C), or[U-13C]glutamine plus unlabeled glutamate (column D), for details see Materials and methods. Results are presented as means +s.d. in nmol/mg protein, and P < 0.05 was considered statistically significant. aDifferent from the corresponding metabolite in the Agroup; bdifferent from the corresponding metabolite in the B group; cdifferent from the corresponding metabolite in the C group.
Glutamate metabolism in cerebellar neuronsE Olstad et al
6
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
As shown in the previous paragraphs, both[U-13C]glutamine and [U-13C]glutamate are wellsuited as precursors for neurotransmitter formationand substrates for neuronal intermediary metabo-lism. However, in the present study [U-13C]gluta-mate gave a higher percent labeling than[U-13C]glutamine in all metabolites, includingGABA. Sonnewald and McKenna (2002) found that[U-13C]glutamate was superior in labeling of aspar-tate, whereas GABA labeling was only observedfrom [U-13C]glutamine, and not from [U-13C]gluta-mate when unlabeled glutamine was present, incortical synaptosomes. Although [U-13C]glutamatewas a better precursor for GABA than [U-13C]gluta-mine in the present study, glutamine was a rela-tively better precursor for GABA than for the othermetabolites investigated, indicating that GABAsynthesis occurs in a separate compartment (seebelow) consistent with findings in cortical synapto-somes (Sonnewald and McKenna, 2002). Wester-gaard et al (1995) showed that in primary culturesof cortical neurons labeling of aspartate, GABA,and [1,2,3-13C]glutamate was very similar from[U-13C]glutamine and [U-13C]glutamate. Thus, notsurprisingly, GABAergic neurons in the cerebellumappear to have a different substrate preference forGABA synthesis than GABAergic neurons in cere-bral cortex. In cultured cortical neurons, TCA cyclemetabolism of the carbon skeleton subsequentlyused in formation of GABA was more pronouncedfrom [U-13C]glutamine than from [U-13C]glutamate(Westergaard et al, 1995). This was also observed inthe present study in cultured cerebellar neurons.With [U-13C]glutamate as the precursor, approxi-mately 60% of the labeled succinate, malate,aspartate, and citrate was uniformly labeled andthus derived from the first turn, B20% was from thesecond and B10% from the third turn in the TCAcycle. When [U-13C]glutamine was present in theexperimental medium, these numbers were B40%,B30%, and B25%, respectively, and similar resultswere seen in GABA. Thus, cycling from glutaminewas more prominent than that from glutamate. Thefact that mitochondrial metabolism of the carbonskeleton of endogenous glutamate derived fromglutamine and glutamate taken up into the cellsdiffered could be due to differences in the distribu-tion of the enzyme phosphate activated glutaminasecatalyzing the formation of glutamate from gluta-mine. This compartmentation could perhaps berelated to intracellular mitochondrial heterogeneity(Westergaard et al, 1995; Sonnewald et al, 1998).Interestingly, label distribution indicating TCAcycling of the carbon skeleton subsequently con-verted into GABA was similar whether [U-13C]glu-tamate or [U-13C]glutamine was the precursorpresent in the experimental medium, showingintercellular compartmentation in addition to theintracellular compartmentation mentioned above.
To evaluate the preference for glutamate andglutamine as substrates for intermediary metabolism
in cultured brain cells or synaptosomes, bothsubstrates have to be present in the mediumsimultaneously. When Sonnewald and McKenna(2002) incubated synaptosomes with [U-13C]gluta-mate in the presence of unlabeled glutamine, thecarbon skeleton from [U-13C]glutamate entered theTCA cycle and labeled aspartate and [1,2,3-13C]glu-tamate, but not GABA. However, label from[U-13C]glutamine in the presence of unlabeledglutamate was incorporated into GABA, but notaspartate. This is consistent with what has beenreported in cortex for the two precursors appliedseparately (Westergaard et al, 1995), showing thatthe compartmentation is maintained under morephysiological conditions with both substrates pre-sent (Sonnewald and McKenna, 2002). The impor-tance of glutamine as a precursor and thus theimportance of the glutamate–glutamine–GABA cy-cle for GABA synthesis is surprising since reuptakeof GABA into the presynapse is believed to beprominent (Schousboe, 2003). Neurotransmitterglutamate, however, is thought to be removed fromthe synaptic cleft mainly by uptake into astrocytes(Schousboe et al, 1977; Danbolt, 2001). On the basisof this, it can be assumed that the importance ofthe glutamate–glutamine cycle should be greater forthe synthesis of neurotransmitter glutamate thanfor GABA. However, results from the present studysuggest otherwise. Consumption of glutamate in thecerebellar neurons was twice that of glutaminewhen given alone and together with glutamine,even though the glutamine concentration in themedium was twice as high as that of glutamate. Mostimportantly, glutamine consumption was reducedby nearly 50% in the presence of glutamatecompared with when glutamate was not present inthe experimental medium. Thus, glutamate couldsubstitute for glutamine, but the reverse was not thecase. Labeling of intracellular metabolites was onlyslightly reduced when neurons were incubated inmedium containing [U-13C]glutamate in the pre-sence of unlabeled glutamine compared with thatin the absence of glutamine. Surprisingly, whenunlabeled glutamate was present together with[U-13C]glutamine, labeling was almost abolishedin all metabolites measured. The carbon skeletonof [U-13C]glutamate and [U-13C]glutamine will, inthe TCA cycle, not only be distributed into themetabolites mentioned above, but also be convertedto 13CO2, which is not detected by the experimentalsetup used in the present study. As mentionedabove, the carbon skeleton of glutamine stayed inthe TCA cycle longer than that of glutamate andthus it is likely that a higher percentage of glutaminecompared with that of glutamate was convertedto 13CO2.
Considering the results presented, it can bepostulated that the glutamate–glutamine cycle is ofless importance for neurons in the cerebellum thanwhat has been described for cerebral corticalneurons. This is further supported by the fact that
Glutamate metabolism in cerebellar neuronsE Olstad et al
7
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
granule neurons are by far the most abundant cellsin the rat cerebellum (265� 106) and outnumber glia(2.2� 106) in the granule layer significantly (Korboet al, 1993). A similar quantitative neuronal dom-inance has been reported in human cerebellum(Andersen et al, 1992). Thus, in the cerebellarcortex, glial processes do not envelop all synapses.Indeed, it has been shown that around synapsesbetween the parallel fibers, the axons of granuleneurons, and interneuron dendrites, astrocyte pro-cesses are lacking (Danbolt, 2001). Therefore, thedistance from the synapse to the nearest glial cell isquite large and, in addition, the relative number ofglutamate transporters on these astrocytes is low(Danbolt, 2001). However, it has been shown that inthe cerebellar granule cell layer, the density ofmRNA for a neuronal glutamate transporter wasvery high (Velaz-Faircloth et al, 1996). Consideringthe relatively large distance between synapticregions of cerebellar granule cells and astrocytesand the high level of glutamate transporters on theseneurons, it can be argued that reuptake of glutamatemust be important for granule neurons. This hasindeed been shown by Waagepetersen et al (2005),who reported that the intracellular pool of glutamatein cerebellar neurons was dependent on reuptake ofextracellular glutamate.
The ‘Partial Tricarboxylic Acid Cycle’
Battaglioli and Martin (1990) showed that insynaptosomes, aspartate synthesis was stronglystimulated by glutamate and glutamine, but thestimulation by glutamate was greatest. Similarly, inthe present study, an increase in intracellularglutamate concentration was always accompaniedby a corresponding increase in that of aspartate.The intracellular glutamate concentration washighest in the cells incubated with both glutamateand glutamine, followed by those incubated withglutamate alone, and thereafter cells incubatedwith glutamine, which also led to an increasecompared with control. The coupling betweenglutamate and aspartate can be explained by thefact that cerebellar neurons have a high activity ofglutamate dehydrogenase and aspartate aminotransferase (Drejer et al, 1985; Westergaard et al,1991; Zaganas et al, 2001). Thus, entry of glutamatevia a-ketoglutarate into the TCA cycle and conver-sion of oxaloacetate to aspartate is very efficientin the cerebellum and energy is obtained from thisso-called ‘partial TCA cycle’ (Hertz et al, 1991;Sonnewald and McKenna, 2002).
GABA Shunt in Cerebellar Neurons
The cerebellar neuronal cultures consist primarilyof glutamatergic granule cells with a minor con-tribution of GABAergic stellate and basket neurons,that is, GABAergic as well as glutamatergic
characteristics are expressed in these cultures(Pearce et al, 1981; Hertz et al, 1985; Hertz andSchousboe, 1987; Drejer and Schousboe, 1989;Kovacs et al, 2003). Thus, cultures of dissociatedcerebellum constitute an excellent model system forthe in vivo situation, in which the association ofGABAergic with glutamatergic neurons can beinvestigated. The major enzyme responsible forGABA synthesis in brain, glutamate decarboxylase,was shown to be present in 6% of culturedcerebellar neurons (Sonnewald et al, 2004) indicat-ing the presence of two distinct cell types and thus,at least two cellular compartments. In the presentstudy, the amount of intracellular GABA in thecultures was increased to the same extent by allexperimental conditions, compared with controlcultures. This is in contrast to the results obtainedfor aspartate, which increased with increasingglutamate concentration, as mentioned above, show-ing cellular compartmentation. It has been shownthat the glutamate decarboxylase positive neurons,which constitute the GABAergic compartment,produce GABA during the whole culture periodand that GABA is distributed throughout the wholeculture (Sonnewald et al, 2006). In the presentstudy, no labeled substrates were present in themedium during the 7-day culture period, thusGABA produced during this time was unlabeled.When medium containing [U-13C]glutamate or[U-13C]glutamine was added to the cultures for the2-h incubation period, labeled GABA could beformed only in the small number of GABAergiccells. Hence, most GABA in the cultures in general,and in the cerebellar granule neurons in particular,was unlabeled at the time of culture extraction.
GABA can be catabolized via the GABA shunt, inwhich the carbon skeleton of GABA enters the TCAcycle after conversion to succinate. Interestingly,percent labeling of succinate was very similar to thatof GABA and much lower than that of the other TCAcycle intermediates measured. The number ofGABAergic neurons in the cultures is small, andthus it seems unlikely that the succinate contentin these neurons could account for the dilution oflabeling in the total succinate pool. It can beassumed that catabolism of GABA and entry ofthe carbon skeleton of GABA into the TCA cyclethrough the GABA shunt is taking place in theglutamatergic cells from unlabeled GABA. Hence, apossible function of GABA in the glutamatergicneurons is the ability to produce a separatesuccinate pool for potential energy production inthe TCA cycle, suggesting a special role for theGABA shunt in the cerebellum.
Conclusion
Through the results presented, it could be shownthat glutamate is preferred over glutamine as asubstrate for intermediary metabolism in cultured
Glutamate metabolism in cerebellar neuronsE Olstad et al
8
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
cerebellar neurons. It can be concluded that theseneurons rely more on reuptake of glutamate thansupply of glutamine from astrocytes for glutamatehomeostasis.
Acknowledgements
We thank Bente Urfjell and Lars Evje for excellenttechnical assistance.
References
Andersen BB, Korbo L, Pakkenberg B (1992) A quantita-tive study of the human cerebellum with unbiasedstereological techniques. J Comp Neurol 326:549–60
Battaglioli G, Martin DL (1990) Stimulation of synaptoso-mal gamma-aminobutyric acid synthesis by glutamateand glutamine. J Neurochem 54:1179–87
Berl S, Clarke D (1983) The metabolic compartmentationconcept. In: Glutamine, glutamate and GABA in thecentral nervous system (Hertz L, Kvamme E, McGeerEG, Schousboe A, eds), New York: Alan R. Liss, Inc.,205–17
Biemann K (1962) Mass spectrometry: Organic chemistryapplications. New York: McGraw-Hill, 223–7
Daikhin Y, Yudkoff M (2000) Compartmentation of brainglutamate metabolism in neurons and glia. J Nutr130:1026S–31S
Dolinska M, Zablocka B, Sonnewald U, Albrecht J (2004)Glutamine uptake and expression of mRNA’s ofglutamine transporting proteins in mouse cerebellarand cerebral cortical astrocytes and neurons. Neuro-chem Int 44:75–81
Drejer J, Larsson OM, Kvamme E, Svenneby G, Hertz L,Schousboe A (1985) Ontogenetic development ofglutamate metabolizing enzymes in cultured cerebellargranule cells and in cerebellum in vivo. Neurochem Res10:49–62
Drejer J, Schousboe A (1989) Selection of a pure cerebellargranule cell culture by kainate treatment. NeurochemRes 14:751–4
Frandsen A, Drejer J, Schousboe A (1989) Direct evidencethat excitotoxicity in cultured neurons is mediated viaN-methyl-D-aspartate (NMDA) as well as non-NMDAreceptors. J Neurochem 53:297–9
Geddes JW, Wood JD (1984) Changes in the amino acidcontent of nerve endings (synaptosomes) induced bydrugs that alter the metabolism of glutamate andgamma-aminobutyric acid. J Neurochem 42:16–24
Grill V, Bjorkman O, Gutniak M, Lindqvist M (1992) Brainuptake and release of amino acids in non diabetic andinsulin-dependent diabetic subjects: important role ofglutamine release for nitrogen balance. Metabolism41:28–32
Hertz L, Juurlink BHJ, Szuchet S (1985) Cell cultures. In:Handbook of neurochemistry (Latja A, ed), New York:Plenum Press, 603–61
Hertz L, Peng L, Westergaard N, Yudkoff M, Schousboe A(1991) Neuronal–astrocytic interactions in metabolismof transmitter amino acids of the glutamate family. In:Drug research related to neuroactive amino acids
(Schousboe A, Diemer NH, Kofod H, eds), Copenhagen,Denmark: Munksgaard, 30–48
Hertz L, Schousboe A (1987) Primary cultures of GABAer-gic and glutamatergic neurons as model systems tostudy neurotransmitter functions. I. Differentiatedcells. In: Model systems of development and aging ofthe nervous system (Vernadakis A, Privat A, Lauder JM,Timiras PS, Giacobini E, eds), Boston: Martinus-Nijhoff, 19–31
Korbo L, Andersen BB, Ladefoged O, Moller A (1993) Totalnumbers of various cell types in rat cerebellar cortexestimated using an unbiased stereological method.Brain Res 609:262–8
Kovacs AD, Cebers G, Cebere A, Liljequist S (2003) Loss ofGABAergic neuronal phenotype in primary cerebellarcultures following blockade of glutamate reuptake.Brain Res 977:209–20
Mawhinney TP, Robinett RS, Atalay A, Madson MA (1986)Analysis of amino acids as their tert-butyldimethylsilylderivatives by gas-liquid chromatography and massspectrometry. J Chromatogr 358:231–42
McKenna MC, Tildon JT, Stevenson JH, Boatright R,Huang S (1993) Regulation of energy metabolism insynaptic terminals and cultured rat brain astrocytes:differences revealed using aminooxyacetate. Dev Neuro-sci 15:320–9
McKenna MC, Tildon JT, Stevenson JH, Hopkins IB (1994)Energy metabolism in cortical synaptic terminals fromweanling and mature rat brain: evidence for multiplecompartments of tricarboxylic acid cycle activity. DevNeurosci 16:291–300
Norenberg MD, Martinez-Hernandez A (1979) Fine struc-tural localization of glutamine synthetase in astrocytesof rat brain. Brain Res 161:303–10
Pearce BR, Currie DN, Beale R, Dutton GR (1981)Potassium-stimulated, calcium-dependent release of[3H]GABA from neuron- and glia-enriched cultures ofcells dissociated from rat cerebellum. Brain Res206:485–9
Peng L, Hertz L, Huang R, Sonnewald U, Petersen SB,Westergaard N, Larsson O, Schousboe A (1993) Utiliza-tion of glutamine and of TCA cycle constituents asprecursors for transmitter glutamate and GABA. DevNeurosci 15:367–77
Qu H, Waagepetersen HS, van HM, Wolt S, Dale O,Unsgard G, Sletvold O, Schousboe A, Sonnewald U(2000) Effects of thiopental on transport and metabo-lism of glutamate in cultured cerebellar granuleneurons. Neurochem Int 37:207–15
Santos SS, Gibson GE, Cooper AJ, Denton TT, ThompsonCM, Bunik VI, Alves PM, Sonnewald U (2006)Inhibitors of the alpha-ketoglutarate dehydrogenasecomplex alter [1-13C]glucose and [U-13C]glutamatemetabolism in cerebellar granule neurons. J NeurosciRes 83:450–8
Schousboe A (2003) Role of astrocytes in the maintenanceand modulation of glutamatergic and GABAergicneurotransmission. Neurochem Res 28:347–52
Schousboe A, Meier E, Drejer J, Hertz L (1989) Preparationof primary cultures of mouse (rat) cerebellar granulecells. In: A Dissection and tissue culture manual of thenervous system (Shahar A, de Vellis J, Vernadakis A,Haber B, eds), New York: Liss, 203–6
Schousboe A, Svenneby G, Hertz L (1977) Uptake andmetabolism of glutamate in astrocytes cultured fromdissociated mouse brain hemispheres. J Neurochem29:999–1005
Glutamate metabolism in cerebellar neuronsE Olstad et al
9
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
Shank RP, Bennett GS, Freytag SO, Campbell GL (1985)Pyruvate carboxylase: an astrocyte-specific enzymeimplicated in the replenishment of amino acid neuro-transmitter pools. Brain Res 329:364–7
Sonnewald U, Hertz L, Schousboe A (1998) Mitochondrialheterogeneity in the brain at the cellular level. J CerebBlood Flow Metab 18:231–7
Sonnewald U, Kortner TM, Qu H, Olstad E, Sunol C,Bak LK, Schousboe A, Waagepetersen HS (2006)Demonstration of extensive GABA synthesis in thesmall population of GAD positive neurons in cerebellarcultures by the use of pharmacological tools. Neuro-chem Int 48:572–8
Sonnewald U, McKenna M (2002) Metabolic compart-mentation in cortical synaptosomes: influence ofglucose and preferential incorporation of endogenousglutamate into GABA. Neurochem Res 27:43–50
Sonnewald U, Olstad E, Qu H, Babot Z, Cristofol R,Sunol C, Schousboe A, Waagepetersen H (2004)First direct demonstration of extensive GABA synthesisin mouse cerebellar neuronal cultures. J Neurochem91:796–803
Sonnewald U, Westergaard N, Petersen SB, Unsgard G,Schousboe A (1993a) Metabolism of [U-13C]gluta-mate in astrocytes studied by 13C NMR spectro-scopy: incorporation of more label into lactatethan into glutamine demonstrates the importanceof the tricarboxylic acid cycle. J Neurochem 61:1179–1182
Sonnewald U, Westergaard N, Schousboe A, Svendsen JS,Unsgard G, Petersen SB (1993b) Direct demonstrationby [13C]NMR spectroscopy that glutamine from astro-cytes is a precursor for GABA synthesis in neurons.Neurochem Int 22:19–29
Sonnewald U, White LR, Odegard E, Westergaard N,Bakken IJ, Aasly J, Unsgard G, Schousboe A (1996)MRS study of glutamate metabolism in culturedneurons/glia. Neurochem Res 21:987–93
Su TZ, Campbell GW, Oxender DL (1997) Glutaminetransport in cerebellar granule cells in culture. BrainRes 757:69–78
van den Berg CJ, Garfinkel D (1971) A stimulation study ofbrain compartments. Metabolism of glutamate andrelated substances in mouse brain. Biochem J123:211–8
Velaz-Faircloth M, McGraw TS, Malandro MS, FremeauRT, Jr, Kilberg MS, Anderson KJ (1996) Characterizationand distribution of the neuronal glutamate transporterEAAC1 in rat brain. Am J Physiol 270:C67–75
Waagepetersen HS, Qu H, Sonnewald U, Shimamoto K,Schousboe A (2005) Role of glutamine and neuronalglutamate uptake in glutamate homeostasis and synth-esis during vesicular release in cultured glutamatergicneurons. Neurochem Int 47:92–102
Westergaard N, Fosmark H, Schousboe A (1991) Metabo-lism and release of glutamate in cerebellar granule cellscocultured with astrocytes from cerebellum or cerebralcortex. J Neurochem 56:59–66
Westergaard N, Sonnewald U, Petersen SB, Schousboe A(1995) Glutamate and glutamine metabolism in cul-tured GABAergic neurons studied by 13C NMR spectro-scopy may indicate compartmentation andmitochondrial heterogeneity. Neurosci Lett 185:24–8
White LR, Garseth M, Aasly J, Sonnewald U (2004)Cerebrospinal fluid from patients with dementiacontains increased amounts of an unknown factor.J Neurosci Res 78:297–301
Yudkoff M, Zaleska MM, Nissim I, Nelson D, Erecinska M(1989) Neuronal glutamine utilization: pathwaysof nitrogen transfer studied with [15N]glutamine.J Neurochem 53:632–40
Zaganas I, Waagepetersen HS, Georgopoulos P, SonnewaldU, Plaitakis A, Schousboe A (2001) Differential expres-sion of glutamate dehydrogenase in cultured neuronsand astrocytes from mouse cerebellum and cerebralcortex. J Neurosci Res 66:909–13
Glutamate metabolism in cerebellar neuronsE Olstad et al
10
Journal of Cerebral Blood Flow & Metabolism (2006), 1–10
Paper 4
Long-term kainic acid exposure reveals compartmentation of
glutamate and glutamine metabolism in cultured cerebellar
neurons
Olstad E, Qu H and Sonnewald U.
Neurochem Int (2006) in press
+ Models
NCI-1967; No of Pages 10
Long-term kainic acid exposure reveals compartmentation of glutamate
and glutamine metabolism in cultured cerebellar neurons
Elisabeth Olstad a,b, Hong Qu c, Ursula Sonnewald a,*a Department of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway
b St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norwayc Centre for Molecular Biology and Neuroscience, Department of Anatomy, University of Oslo, Norway
Received 15 September 2006; received in revised form 22 October 2006; accepted 6 November 2006
Abstract
Glutamate neurotoxicity is implicated in most neurodegenerative diseases, and in the present study the long-term effects of the glutamate
agonist kainic acid (KA) on cerebellar neurons are investigated. Primary cell cultures, mainly consisting of glutamatergic granule neurons, were
cultured in medium containing 0.05 or 0.50 mM KA for 7 days and subsequently incubated in medium containing [U-13C]glutamate or
[U-13C]glutamine. The amount of protein and number of cells were greatly reduced in cultures exposed to 0.50 mM KA compared to those exposed
to 0.05 mM KA. Glutamine consumption was not affected by KA concentration, whereas that of glutamate was decreased by high KA, confirming
reduction in glutamate transport reported earlier. Neurons cultured with 0.50 mM KA and incubated with glutamate contained decreased amounts
of glutamate, aspartate and GABA compared to those cultured with 0.05 mM KA. Incubation of cells exposed to 0.50 mM KA with glutamine led
to an increased amount of glutamate compared to cells exposed to 0.05 mM KA, whereas the intracellular amounts of aspartate and GABA
remained unaffected by KA concentration. Furthermore, mitochondrial metabolism of a-[U-13C]ketoglutarate derived from [U-13C]glutamate and
[U-13C]glutamine was significantly reduced by 0.50 mM KA. The results presented illustrate differential vulnerability to KA and can only be
understood in terms of inter- and intracellular compartmentation.
Fig. 2. The amount of protein in cultures (A) and photomicrographs (B) of cerebellar neurons cultured in medium containing 0.05 or 0.50 mM kainic acid (KA) in
35 mm Petri dishes. On day 7 in vitro, the culture medium was removed, and the cells were incubated for 2 h in medium containing either no additions, 0.25 mM
[U-13C]glutamate or 0.50 mM [U-13C]glutamine, for details see Section 2. The protein amount was not affected by different incubation conditions, thus cultures from
different incubation conditions are presented together. Results are mean � S.D. in mg protein per culture (n = 12 in each group), and p < 0.05 was considered
statistically significant. Bars in photomicrographs represent 0.100 mm. *Different from the group cultured in medium containing 0.05 mM KA.
Fig. 3. Cellular content of glutamate, aspartate, glutamine and GABA detected by HPLC in extracts of cultured cerebellar neurons. Cells were cultured in medium
containing either 0.05 or 0.50 mM kainic acid (KA). On day 7 in vitro, the culture medium was removed, and the cells were incubated for 2 h in medium with either no
additions (ctr, n = 3), 0.25 mM [U-13C]glutamate (glu, n = 6), 0.50 mM [U-13C]glutamine (gln, n = 6), for details see Section 2. Results are presented as mean � S.D.
in nmol/mg protein, and p < 0.05 was considered statistically significant. aDifferent from the ctr group; bDifferent from the glu group; *Different from the
corresponding group cultured in medium containing 0.05 mM KA.
E. Olstad et al. / Neurochemistry International xxx (2006) xxx–xxx 5
+ Models
NCI-1967; No of Pages 10
Please cite this article in press as: Olstad, E. et al., Long-term kainic acid exposure reveals compartmentation of glutamate and glutamine
metabolism in cultured cerebellar neurons, Neurochem. Int. (2006), doi:10.1016/j.neuint.2006.11.004
Fig. 4. Distribution of 13C label in glutamate, aspartate and GABA detected by mass spectrometry analysis of cell extracts of cultured cerebellar neurons after
incubation with 0.25 mM [U-13C]glutamate (A, n = 6) or 0.50 mM [U-13C]glutamine (B, n = 6). Cells were cultured in medium containing either 0.05 mM or
0.50 mM kainic acid (KA), for details see Section 2. Note that uniformly labeled glutamate and GABA is presented, and that GABA from the second and third turn is
presented together due to the fact that the same isotopomers arise from both turns. Results are presented as mean � S.D. in percent of the total labeled compound, and
p < 0.05 was considered statistically significant. *Different from the corresponding group cultured in medium containing 0.05 mM KA.
E. Olstad et al. / Neurochemistry International xxx (2006) xxx–xxx6
+ Models
NCI-1967; No of Pages 10
compared to cells cultured in the presence of 0.05 mM KA.
Incubation in medium with [U-13C]glutamine led to similar
intracellular GABA concentrations regardless of KA exposure
during the culture period.
The cells’ consumption of [U-13C]glutamate or [U-13C]glu-
tamine was determined by quantifying the amounts of the two
precursors in the incubation media and subtracting these from
the amounts added. Cultures exposed to 0.50 mM KA and
incubated in medium containing [U-13C]glutamate, consumed
approximately half of the amount of glutamate compared to
cultures exposed to 0.05 mM KA, 734 � 184 nmol/mg protein
versus 1537 � 55 nmol/mg protein during the 2 h incubation,
respectively. In contrast, glutamine consumption by neurons
incubated in medium containing [U-13C]glutamine was not
affected by KA concentration. The consumption of glutamine
was 719 � 59 nmol/mg protein in the low KA group and
627 � 162 nmol/mg protein per 2 h in the high KA group.
3.3. 13C labeling of metabolites
In Table 1, percent 13C enrichment in glutamate, aspartate
and GABA in cell extracts after incubation in medium with
[U-13C]glutamate or [U-13C]glutamine is presented. In general,
Please cite this article in press as: Olstad, E. et al., Long-term kainic a
metabolism in cultured cerebellar neurons, Neurochem. Int. (2006), do
in glutamate and aspartate, percent enrichment after incubation
in the presence of [U-13C]glutamate was very high (>85%),
and higher than after incubation in medium with [U-13C]glu-
tamine (approximately 45–65%). In GABA, percent enrich-
ment was much lower, but also in this metabolite, the
enrichment was higher after incubation in medium with
[U-13C]glutamate than with [U-13C]glutamine, �30% and
�20%, respectively. Cells cultured in the presence of 0.50 mM
KA and incubated in medium containing [U-13C]glutamate had
a higher percent enrichment in glutamate and aspartate, but not
in GABA, compared to cells cultured in the presence of
0.05 mM KA and incubated in medium with [U-13C]glutamate.
On the other hand, cells exposed to 0.50 mM KA and incubated
in the presence of [U-13C]glutamine, had a higher percent
enrichment in all metabolites than cells cultured in medium
containing 0.05 mM KA and incubated in medium with
[U-13C]glutamine.
In order to investigate the effect of KA on metabolism, 13C
labeling from [U-13C]glutamate and [U-13C]glutamine arising
from different turns in the TCA cycle was calculated. In Fig. 4
percent labeling from [U-13C]glutamate and [U-13C]glutamine
in uniformly labeled glutamate and GABA and also glutamate,
aspartate and GABA from precursors derived from the three
cid exposure reveals compartmentation of glutamate and glutamine
Pyruvate recycling in cultured neurons from cerebellum
Olstad E, Olsen GM, Qu H and Sonnewald U.
J Neurosci Res (2006) in press
Pyruvate Recycling in Cultured NeuronsFrom Cerebellum
Elisabeth Olstad,1,2 Grethe M. Olsen,1 Hong Qu,3 and Ursula Sonnewald1*1Department of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway2St. Olavs Hospital, Trondheim, Norway3Centre for Molecular Biology and Neuroscience, Department of Anatomy, University of Oslo,Oslo, Norway
Pyruvate recycling is a pathway for complete oxidation ofglutamate and cellular location, and the physiological sig-nificance of such recycling has been debated during thelast decade. The present study was aimed at elucidatingwhether recycling takes place in neuron-enriched culturesof dissociated cerebella, consisting mainly of glutamater-gic granule cells, some GABAergic neurons, and fewastrocytes. These cultures and cultures of astrocytesfrom cerebellum were incubated in medium containing[U-13C]glutamate, and cell extracts were analyzed by gaschromatography and mass spectrometry. Additionally, inthe case of the neurons, a magnetic resonance (MR)spectrum was obtained. It could be shown that the atompercentage excess of the isotopomer representing pyru-vate recycling in glutamate (M + 4) was similar for astro-cytes and neuron-enriched cultures. However, the lattershowed more recycling in glutamine (synthesized in thesmall fraction of astrocytes) than the pure astrocyte cul-tures, whereas the reverse was the case for aspartate. Infact, the atom percentage excess of the isotopomer rep-resenting pyruvate recycling in glutamine was slightly butsignificantly higher than that in glutamate in the neuron-enriched cultures. It can be concluded that pyruvate recy-cling is clearly present in neurons, and this was verifiedby MR spectroscopy. VVC 2007 Wiley-Liss, Inc.
Pyruvate carboxylase located in astrocytes is thoughtto be the enzyme responsible for anaplerosis in brain(Patel, 1974), and carboxylation of pyruvate has beendemonstrated in rat, mouse, and human brain (Lapidotand Gopher, 1994; Hassel et al., 1995; Oz et al., 2004;Patel et al., 2005; Melø et al., 2006). Previous experimentsand results from cell cultures incubated in the presence of[U-13C]glucose and 3-nitropropionic acid confirmed thatpyruvate carboxylation takes place in astrocytes but notneurons (Yu et al., 1983; Shank et al., 1985; Waagepe-tersen et al., 2001; Qu et al., 2001). During developmentanaplerosis (filling up) is necessary, because concentrationsof glutamate and glutamine in brain increase (Tkae et al.,2003), whereas in adults anaplerosis is not self-evident. It isgenerally accepted that the adult brain has to replenish the
tricarboxylic acid (TCA) cycle when a four (or more) car-bon unit such as glutamine or a lactate molecule derivedfrom TCA cycle intermediates (malate or oxaloacetate)leaves the brain. Glutamine is indeed released from thebrain (Grill et al., 1992), but it has not been shown thatlactate from brain is TCA cycle derived. Another possibil-ity is that a four (or more) carbon unit is degraded in thebrain. Pyruvate recycling is such a pathway in which com-pounds such as glutamate, glutamine, or aspartate, whichare originally derived from pyruvate carboxylation, can bedegraded to pyruvate and reenter the TCA cycle as acetylCoA (see Fig. F11). Previous studies have shown that pyru-vate recycling takes place in rat brain, and initially the cel-lular location was thought to be neurons (Cerdan et al.,1990). However, cell culture studies pointed toward astro-cytes as the site for recycling (Sonnewald et al., 1996a;Bakken et al., 1997a,b, 1998a; Haberg et al., 1998; Alveset al., 2000; Waagepetersen et al., 2002).
The present study was undertaken to reinvestigatethe cellular location of pyruvate recycling. By using 13C la-beled compounds and 13C magnetic resonance spectros-copy (MRS), it is possible to monitor cellular metabolismand astrocyte–neuron interactions. Various 13C-labeledsubstrates have been used to unravel different aspects ofcerebral metabolism. We incubated cerebellar neurons andastrocytes with medium containing [U-13C]glutamate.Analysis of neuronal cell extracts by MRS and the moresensitive method gas chromatography/mass spectrometry(GC/MS) revealed that pyruvate recycling takes place inneurons as well as in astrocytes.
MATERIALS AND METHODS
Materials
NMRI mice were obtained from Taconic M&B (Co-penhagen, Denmark). Plastic tissue culture dishes were pur-
Received 29 September 2006; Revised 21 November 2006; Accepted 28
November 2006
Published online 00 Month 2007 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/jnr.21208
Journal of Neuroscience Research 85:000–000 (2007)
' 2007 Wiley-Liss, Inc.
chased from Nunc A/S (Roskilde, Denmark) and fetal calf se-rum (FCS) from Seralab Ltd. (Sussex, United Kingdom).Culture medium and glutamate receptor antagonists DNQX(6,7-dinitroquinoxaline-2,3-dione) and D-AP5 (D-2-amino-5-phosphonopentanoic acid) were from Sigma (St. Louis,MO). [U-13C]glutamate (98%+ enriched) and 99.9% D2Owere from Cambridge Isotope Laboratories (Woburn, MA);ethylene glycol was from Merck (Darmstadt, Germany), andthe GC/MS derivatization reagent MTBSTFA (N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide) + 1% t-BDMS-Cl(tert-butyldimethylchlorosilane) was purchased from RegisTechnologies, Inc. (Morton Grove, IL). All other chemicalswere of the purest grade available from regular commercialsources.
Cell Cultures
Neuron-enriched cerebellar cultures were isolated andcultured from 7-day-old mice as described by Schousboe et al.(1989). Briefly, the tissue was trypsinized, followed by tritura-tion in a DNase solution containing a trypsin inhibitor fromsoybeans. Cells were suspended (2.75 3 106 cells/ml) in amodified Dulbecco’s minimum essential medium (DMEM;Hertz et al., 1982), containing 24.5 mM KCl, 31 mM glu-cose, 7 lM p-aminobenzoic acid, 0.05 mM kainic acid, and
10% (v/v) fetal calf serum (FCS) and seeded in poly-D-lysine-coated Petri dishes (2 ml/35 mm). After 48 hr in culture,20 lM (final concentration) cytosine arabinoside was added tothe medium to prevent astrocytic proliferation. Experimentswere performed on 7-day-old cultures.
Cerebellar astrocytes were cultured as described byHertz et al. (1989). Briefly, cerebellum was taken from 7-day-old mice and passed through Nitex nylon netting (80-lmpore size) into DMEM containing 20% (v/v) FCS. Mediumwas changed 2 days after plating and subsequently twice perweek, gradually changing to 10% FCS. From the third week,dibutyryl-cAMP was added to the medium to promote mor-phological differentiation of the astrocytes. Experiments wereperformed on 3-week-old cultures.
Experiments Using [U-13C]Glutamate for MRS andGC/MS Analysis
The culture medium was removed, and the cells wereincubated for 2 hr at 378C in serum-free DMEM (preparedwithout glutamine) containing 3 mM glucose and [U-13 C]glutamate (neurons, 0.25 mM; astrocytes 0.5 mM). To avoidthe toxicity of glutamate to neurons during the incubation,two glutamate receptor antagonists, DNQX (25 lM), an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)/kainate-selective antagonist, and D-AP5 (100 lM),an N-methyl-D-aspartate (NMDA) antagonist, were alsopresent in the incubation medium of neurons (Frandsenet al., 1989). After the incubation period, the cells werewashed twice with cold phosphate-buffered saline andextracted with 70% (v/v) ethanol. The cell extracts werescraped off the dishes and centrifuged at 10,000g for 15 minto separate the metabolites from the insoluble proteins. Thesupernatants (cell extracts) were divided into two parts; onewas lyophilized for subsequent sample preparation for GC/MS analyses (six samples), and the remaining halves werepooled and lyophilized (one sample) for MR spectroscopy(neurons).
GC/MS
Lyophilized cell extracts were redissolved in HCl (10 mM),adjusted to pH <2 with 6 M HCl, and dried under atmos-pheric air. The amino acids were extracted into an organicphase of ethanol and benzene and dried again under atmos-pheric air before derivatization with MTBSTFA in the pres-ence of 1% t-BDMS-Cl (Mawhinney et al., 1986). The sam-ples were analyzed on a Hewlett Packard 5890 Series II gaschromatograph linked to a Hewlett Packard 5972 Series massspectrometer.
MR Spectroscopy
The lyophilized sample consisting of six pooled cellextracts of neuron-enriched cultures was redissolved in D2O.A proton decoupled 150.92-MHz 13C MR spectrum wasobtained on a Bruker 600 spectrometer using a Bruker Bio-Spin CryoProbe (Bruker Analytik GmbH, Rheinstetten, Ger-many). The spectrum was accumulated using a 308 pulseangle, an acquisition time of 1.08 sec, and a 0.5-sec relaxationdelay. The number of scans was 129,024.
Fig. 1. Schematic presentation of pyruvate recycling from glutamate,which can be amidated to glutamine or enter the TCA cycle as a-ketoglutarate and be converted to uniformly labeled succinate and af-ter several steps malate (MAL) and oxaloacetate (OAA). The lattercan further be transaminated into aspartate. If pyruvate recycling isactive, malate via malic enzyme or oxaloacetate via phosphoenolpyr-uvate carboxykinase plus pyruvate kinase can be converted to pyru-vate, which can enter the TCA cycle via acetyl CoA and give rise tounique labeling patterns in glutamate and aspartate.
2 Olstad et al.
Journal of Neuroscience Research DOI 10.1002/jnr
Data Analysis
Atom percentage excess (13C) of glutamate, glutamine,and aspartate was determined after calibration with unlabeledstandard solutions (Biemann, 1962). In the following, thesums of M + 1, M + 2, and M + 3 in glutamate or glutamineand that of M + 1, M + 2, and M + 4 in aspartate are usedto describe TCA cycle activity, because these isotopomers arederived from the TCA cycle using unlabeled acetyl CoA. Todescribe pyruvate recycling, which is also part of TCA cycleactivity, M + 4 in glutamate or glutamine and M + 3 inaspartate are used. It should be noted that this is a simplifica-tion in which the pyruvate recycling pathway is underesti-mated and the TCA cycle involving unlabeled acetyl CoA isoverestimated (see Figs.F2,F3 2, 3). The pyruvate recycling overTCA cycle activity (PR/TCA) ratio was calculated by divid-
ing atom percentage excess for glutamate or glutamine M + 4(aspartate, M + 3) by the sum of TCA cycle derived iso-topomers M + 1, M + 2, and M + 3 (aspartate, the sum ofM + 1, M + 2, and M + 4). Results are presented as means6 SD. Differences between astrocytes and neurons were ana-lyzed statistically by Student’s t-test and differences in atompercentage excess of the corresponding isotopomers betweenglutamate, glutamine, and aspartate with one-way ANOVAfollowed by the LSD (least significant difference) post hoctest. P < 0.05 was considered statistically significant.
RESULTS
[U-13C]glutamate present in the incubation me-dium can enter cells through specific transporter pro-teins. Once inside the cells, [U-13C]glutamate has several
Fig. 2. Schematic representation of possible isotopomers of glutamate and glutamine arising from[U-13C]glutamate via the first turn in the TCA cycle (top) and pyruvate recycling (bottom): solidovals represent 13C, and open ovals represent 12C atoms. The masses (M, M + 1, M + 2, etc.) ofthe different isotopomers are indicated, and the M + 4 isotopomers, which can only result frompyruvate recycling, are shown in boldface.
Pyruvate Recycling in Neurons 3
Journal of Neuroscience Research DOI 10.1002/jnr
metabolic fates. Of interest for pyruvate recycling is theconversion into a-[U-13C]ketoglutarate, which can enterthe TCA cycle to be converted to uniformly labeledsuccinate and after several steps malate and oxaloacetate(OAA). The latter can also be transaminated to aspartate(Fig. 1). [U-13C]oxaloacetate can stay in the cycle andcondense with acetyl CoA to give rise, after severalsteps, to particular labeling patterns in glutamate and glu-tamine (Fig. 2) and aspartate (Fig. 3). However, if pyru-vate recycling is active, malate via malic enzyme orOAA via phosphoenolpyruvate carboxykinase plus pyru-vate kinase can be converted to pyruvate, which canenter the TCA cycle via acetyl CoA (Fig. 1) and give rise
to unique labeling patterns in glutamate (Fig. 2, bottom)and aspartate (Fig. 3, bottom). It should be noted that uni-formly labeled glutamate is formed when [U-13C]OAAcondenses with [1,2-13C]acetyl CoA, and this isotopomercannot be distinguished from the [U-13C]glutamate addedto the medium.
With GC/MS, information about the percentagedistribution of isotopomers with different masses (M, M +1, M + 2, etc.) of each metabolite is obtained. However,the position of the 13C atoms in the isotopomers is notreadily available. From Figures 2 and 3, it can be seen thatM + 4 in glutamate and glutamine and M + 3 in aspartateare the only isotopomer masses resulting from pyruvate
Fig. 3. Schematic representation of possible isotopomers of aspartate arising from [U-13C]glutamatevia the first turn in the TCA cycle (top) and pyruvate recycling (bottom): solid ovals represent13C, and open ovals represent 12C atoms. The masses (M, M + 1, M + 2, etc.) of the differentisotopomers are indicated, and the M + 3 isotopomers, which can only result from pyruvate recy-cling, are shown in boldface.
4 Olstad et al.
Journal of Neuroscience Research DOI 10.1002/jnr
recycling that can be distinguished from those from TCAcycling by GC/MS. By using this method, 13C labelingwas detected in glutamate, glutamine, GABA, aspartate,succinate, fumarate, malate, and citrate in neurons andastrocytes. For simplicity, only glutamate, glutamine, andaspartate labelings are shown, because they illustrate thepoint of this paper clearly. Glutamine (synthesized inastrocytes only) was detected both in the astrocytes and inthe neuron-enriched cultures. In TableT1 I, the percentageuniformly labeled glutamate and glutamine in neurons andastrocytes is presented. The fraction of uniformly labeledglutamate was higher in astrocytes than in neurons,whereas the opposite was the case for glutamine. Further-more, the isotopomers derived from the TCA cycle usingunlabeled acetyl CoA and those derived from pyruvaterecycling in glutamate, aspartate, and glutamine are pre-sented in Table I. It should be noted that some isotopom-ers from pyruvate recycling will also appear with the samenumber of 13C atoms as those derived from the TCA cycle(see Figs. 2, 3). In addition, uniformly labeled isotopomerscan also result from recycling. Therefore, the extent of py-ruvate recycling is underestimated in Table I, insofar asthis pathway is represented only by the M + 4 isotopomerin glutamate and glutamine and by M + 3 in aspartate.From Table I it is clear that atom percentage excess of theisotopomer from the TCA cycle is higher in neurons thanastrocytes for all three amino acids, whereas pyruvate recy-cling in astrocytes and neuron-enriched cultures were sim-ilar in glutamate but not in glutamine and aspartate. Theneuron-enriched cultures showed more atom percentageexcess in the isotopomer derived from recycling in gluta-mine (synthesized in the small fraction of astrocytes) thanin pure astrocyte cultures, whereas the reverse was the casefor aspartate. In fact, the percentage isotopomer from recy-cling in glutamine was slightly but significantly higherthan that in glutamate in the neuron-enriched cultures andlower in the astrocytes.
To visualize pyruvate recycling in a representativeway, the ratio of M + 4 (PR) over the sum of M + 1,M + 2, and M + 3 (TCA) for glutamate and glutaminewas calculated and is presented in parentheses in Table I.For aspartate, the uniformly labeled isotopomer resultingfrom the first TCA cycle turn (Fig. 3) is included in thisratio [(M + 3)/S(M + 1, M + 2 and M + 4)]. Approxi-mately half of the aspartate in the cells (49% 6 9% astro-cytes; 51% 6 1% neurons) was uniformly labeled. Inevaluating the extent to which glutamate, glutamine,and aspartate were derived from pyruvate recycling com-pared with the extent to which isotopomers werederived from TCA cycle activity (PR/TCA ratio), it isclear that pyruvate recycling was more prominent inglutamate in astrocytes than in neurons (Table I). Asmentioned, uniformly labeled aspartate was included inthe calculation of PR/TCA in Table I, and this makes itevident that pyruvate recycling was much smaller thanTCA cycle activity in this metabolite. Furthermore, thePR/TCA ratio for aspartate was much lower than theratios for glutamate and glutamine in neurons, and lowerthan the ratio of glutamate in astrocytes (Table I). ThePR/TCA ratio for glutamine was similar to that of glu-tamate in the astrocytes and similar in pure astrocyte cul-tures and in astrocytes present in neuron-enriched cul-tures.
With 13C MR spectroscopy, it is possible to detectthe position in which a particular compound is 13C labeledin contrast to GC/MS, where only the extent of labelingis detectable under the conditions used in the presentstudy. 13C-labeled glutamate and aspartate were observedin the MR spectrum of extracts from neurons incubatedwith medium containing [U-13C]glutamate (Fig. F44). Toshow enough detail, only the parts of the spectrum thatcontain glutamate C-4 and aspartate C-3 are shown.Looking at the glutamate C-4 frequency range presentedin Figure 4A, peaks showing 13C labeling in the C-4 posi-
*In parenthesis, the ratio of isotopomers derived from pyruvate recycling over those derived from TCA using unlabeled acetyl CoA is calculated for
glutamate, aspartate, and glutamine in cultured neurons and astrocytes from cerebellum. Neuron-enriched and astrocyte cultures from cerebellum were
incubated for 2 hr in medium containing 3 mM glucose, 25 lM DNQX, and 100 lM D-AP5 and 0.25 mM [U-13C]glutamate (neurons, n ¼ 6) or
0.5 mM (astrocytes, n ¼ 6) [U-13C]glutamate; for details, see Materials and Methods. The results are from GC/MS analyses, where [U-13C] is repre-
sented by M + 5 (glutamate and glutamine), isotopomers derived from the TCA cycle using unlabeled acetyl CoA (TCA) by S(M + 1, M + 2 and
M + 3) for glutamate and glutamine and S(M + 1, M + 2 and M + 4) for aspartate, and finally isotopomers from the TCA cycle involving pyruvate
recycling are represented by M + 4 (glutamate and glutamine) and M + 3 (aspartate). Results are presented as means 6 SD, and P < 0.05 was consid-
ered statistically significant.aDifferent from the corresponding group in neurons.bDifferent from atom percent excess of the corresponding glutamate isotopomers in the same cell type.cDifferent from atom percent excess of the corresponding aspartate isotopomers in the same cell type.
Pyruvate Recycling in Neurons 5
Journal of Neuroscience Research DOI 10.1002/jnr
tion also give information about neighboring positions C-3 and C-5, but not about the C-1 and C-2 positions. Thepeaks consist of a doublet of doublets (indicated by arrows)representing [3,4,5-13C]glutamate, one doublet with 52 Hz
splitting from [4,5-13C]glutamate, another doublet with34 Hz splitting from [3,4-13C]glutamate, and finally a sin-glet representing [4-13C]glutamate. Both doublets and thesinglet arise from pyruvate recycling and are indicated byasterisks. The aspartate C-3 peaks (Fig. 4B) show a similarpattern with a doublet of doublets (arrows) representing[2,3,4-13C]aspartate, one doublet with 51 Hz splittingrepresenting [3,4-13C]aspartate arising from TCA cycling(number sign), another doublet with 36 Hz splitting from[2,3-13C]aspartate derived from pyruvate recycling (aster-isks), and finally the [3-13C]aspartate singlet from TCAcycling (number sign). [3,4,5-13C]glutamate seen in thespectrum can be part of the precursor, [U-13C]glutamate,whereas [2,3,4-13C]aspartate can also be included in[U-13C]aspartate derived from [U-13C]glutamate via thefirst turn of the TCA cycle (Fig. 3). [3,4-13C]aspartateand [3-13C]aspartate are derived from the second andthird turns of the TCA cycle, respectively (Fig. 3).[4,5-13C]-, [3,4-13C]-, and [4-13C]glutamate are allderived from pyruvate recycling (Fig. 2). MR spectra ofastrocyte extracts were not obtained; they have been pub-lished previously and showed recycling in glutamate andaspartate (Haberg et al., 1998).
DISCUSSION
Pyruvate recycling is a well-known metabolic path-way in the liver, but it was first detected in brain byCerdan et al. (1990). In a later study by the same authors,the metabolism of [1,2-13C]glucose and [U-13C]-3-hydroxybutyrate was investigated in rat brain via ex vivo13C MR spectroscopy, taking advantage, in particular, ofhomonuclear 13C-13C spin coupling patterns. A quantita-tive analysis of the 13C spectra demonstrated a cerebralpyruvate recycling system contributing maximally 17% ofthe pyruvate metabolism through the pyruvate dehydro-genase in brain (Kunnecke et al., 1993). This recyclingwas believed to take place in neurons and not astrocytes,because it was apparent in glutamate but not in gluta-mine. In 1995, Hassel and Sonnewald reported partialpyruvate recycling in mouse brain astrocytes, because lac-tate formation from the TCA cycle was detected from[2-13C]acetate but not [1-13C]glucose. The enrichmentof total brain lactate from [2-13C]acetate reached approxi-mately 1% in both the C-2 and the C-3 positions infasted mice. It was calculated that this could account for20% of the lactate formed in the glial compartment(Hassel and Sonnewald, 1995). Pyruvate recycling inbrain was also studied in fasted rats receiving either an in-traperitoneal or a subcutaneous injection of [1,2-13C]ace-tate (Haberg et al., 1998). MR spectroscopic analysis ofplasma showed larger amounts of [1, 2-13C]acetate in theintraperitoneal group compared with the subcutaneousgroup, and this was coupled to more pyruvate recyclingdetected in the former group in glutamate and GABA.However, Lapidot and Gopher (1994) were not able todetect such recycling in rabbit brain.
Pyruvate recycling is a pathway for complete oxi-dation of glutamate in the TCA cycle. In cultured corti-
Fig. 4. Parts of the MR spectrum showing glutamate C-4 (A) andaspartate C-3 (B) and the isotopomers responsible for the configura-tion of the peaks: solid ovals represent 13C, and open ovals represent12C atoms. Both peaks consist of a doublet of doublets (indicated byarrows) representing [3,4,5-13C]glutamate and [2,3,4-13C]aspartate,one doublet with approximately 50 Hz splitting from [4,5-13C]gluta-mate (recycling, *) and [3,4-13C]aspartate (TCA cycling, #), anotherdoublet with approximately 35 Hz splitting from [3,4-13C]glutamate(recycling, *) and [2,3-13C]aspartate (recycling, *), and finally a sin-glet representing [4-13C]glutamate (recycling, *) and [3-13C]aspartate(TCA cycling, #). No information can be obtained about labeling inthe C-1 and C-2 positions for glutamate and the C-1 position foraspartate indicated by dashed ovals.
6 Olstad et al.
Journal of Neuroscience Research DOI 10.1002/jnr
cal astrocytes, pyruvate formed from [U-13C]glutamatewas shown to reenter the TCA cycle after conversion toacetyl CoA, as demonstrated by the labeling patterns inaspartate C-2 and C-3, lactate C-2, and glutamate C-4,which provided evidence for pyruvate recycling in astro-cytes (Haberg et al., 1998). Also, in neuron-enrichedcultures from cerebellum, labeling of lactate was detectedfrom [U-13C]glutamate and [U-13C]aspartate, but 13Clabel was not shown to reenter the TCA cycle throughacetyl CoA in these cells (Sonnewald et al., 1996b;Bakken et al., 1998a). Incubating cortical astrocyte cul-tures with [U-13C]glutamine in the presence of gluta-mate or [U-13C]aspartate also led to a small amount ofpyruvate recycling in glutamate (Sonnewald et al.,1996a; Bakken et al., 1997b, 1998a). Recycling was alsodemonstrated in astrocytes using [3-13C]glutamate butcould not be shown in cortical GABAergic neurons(Waagepetersen et al., 2002). Hypoglycemia, a conditionin which acetyl CoA production is reduced, could possi-bly lead to increased pyruvate recycling. However, recy-cling was abolished in astrocytes under this condition(Bakken et al., 1998b). It was hypothesized that cocul-tures of astrocytes and neurons could possibly haveincreased pyruvate recycling compared with astrocytesbecause of transfer of lactate produced by astrocytic mi-tochondria to neurons for glutamate synthesis. Surpris-ingly, experiments with [3-13C]glutamate and neocorti-cal cocultures did not show signs of pyruvate recyclingeven though astrocytes were present (Waagepetersenet al., 2002). However, recycling could be shown whencerebellar cocultures were superfused with mediumcontaining [U-13C]lactate or [U-13C]glucose (Bak et al.,2006).
In the present study, cerebellar astrocytes wereincubated with [U-13C]glutamate, and cell extracts wereanalyzed by GC/MS. As expected, pyruvate recyclingwas detected in glutamate and aspartate. Recycling inaspartate was about twice as high as that evident in glu-tamate. This is due to the fact that [U-13C]glutamatewas added to the medium and constituted 63% of allglutamate in the astrocytes. The sum of the isotopomersfrom the TCA cycle and recycling was 24% of total glu-tamate (Table I). This was the case for 79% (Table I) ofaspartate. Glutamine labeling via pyruvate recycling inastrocytes was much lower than that observed in gluta-mate. This was, however, due not to a lower ratio forpyruvate recycling/cycling in the TCA cycle but to thefact that, under the chosen incubation conditions, only12% of glutamine was derived from mitochondrial me-tabolism compared with 24% of glutamate. The smallpercentage of glutamine derived from TCA cycle activ-ity in astrocytes could explain why Cerdan et al. (1990)suggested that recycling took place in neurons, not astro-cytes.
Neuron-enriched cultures from cerebellum are anexcellent model for studying cerebellar metabolism,because they consist of a majority of glutamatergic withabout 5% GABAergic neurons, mimicking the distribu-tion in brain (Sonnewald et al., 2004a, 2006; Olstad
et al., 2006), and also astrocytes are present, though onlyvery few. When these cultures were incubated in me-dium containing [U-13C]glutamate, GC/MS analysisclearly showed pyruvate recycling in glutamate, aspartate,and glutamine. Furthermore, atom percentage excess ofthe isotopomer derived from pyruvate recycling in gluta-mate in astrocytes and neuron-enriched cultures wassimilar, thereby ruling out that the few astrocytes presentin the neuron-enriched cultures were responsible for therecycling. When evaluating the extent to which gluta-mate was derived from pyruvate recycling comparedwith the extent to which isotopomers were derivedfrom TCA cycle activity using unlabeled acetyl CoA, itis clear that pyruvate recycling was more prominent inglutamate in astrocytes than in neurons. However, this isdue to the fact that in astrocytes less glutamate is derivedfrom the TCA cycle than in neurons, whereas the per-centage excess 13C labeling for glutamate derived frompyruvate recycling (M + 4) was similar in astrocytes andneurons.
Aspartate showed a very low pyruvate recyclingover TCA cycle activity ratio when all TCA-cycle-derived isotopomers, including uniformly labeled aspar-tate, were considered. However, comparing the atompercentage excess of the aspartate isotopomer derivedfrom recycling and that of glutamate and glutamine,aspartate recycling was highest in both cell types.
The pyruvate recycling over TCA cycle activityratio in glutamine (which is only synthesized in astro-cytes) was similar to that in glutamate in the astrocytesand similar in pure astrocyte cultures and in neuron-enriched cultures containing some astrocytes. How-ever, labeling from TCA cycle activity using unlabeledacetyl CoA was more pronounced in glutamineobserved in neuron-enriched than in pure astrocytecultures. Thus, there was more pyruvate recycling inglutamine from neuron-enriched than from astrocyticcultures. In fact, the atom percentage excess of the iso-topomer derived from recycling in glutamine in theneuron-enriched cultures was slightly but significantlyhigher than that in glutamate in the same cell type.This could indicate that glutamate metabolism wascompartmentalized in these neurons, as previouslyshown in various cell types (Waagepetersen et al.,1999; Qu et al., 1999, 2005; Eloqayli et al., 2002;Sonnewald et al., 2004b), and that glutamate derivedfrom recycling was preferentially released from neuronsin the neuron-enriched cultures to be converted toglutamine in the astrocytes. Alternatively, astrocytesmight have a different metabolism in the presence ofneurons than in a monoculture.
To verify pyruvate recycling in neurons, an MRspectrum was obtained of cell extracts, and recycling wasclearly detectable in glutamate and aspartate. The reasonwhy such recycling in neurons has not been detectedearlier could be the enhanced sensitivity of the cryo-MR probe used in the present experiment. Taken to-gether, these results clearly show significant pyruvaterecycling in cerebellar neurons.
Dissertations at the Faculty of Medicine, NTNU 1977 1. Knut Joachim Berg: EFFECT OF ACETYLSALICYLIC ACID ON RENAL FUNCTION 2. Karl Erik Viken and Arne Ødegaard: STUDIES ON HUMAN MONOCYTES CULTURED IN
VITRO 1978 3. Karel Bjørn Cyvin: CONGENITAL DISLOCATION OF THE HIP JOINT. 4. Alf O. Brubakk: METHODS FOR STUDYING FLOW DYNAMICS IN THE LEFT
VENTRICLE AND THE AORTA IN MAN. 1979 5. Geirmund Unsgaard: CYTOSTATIC AND IMMUNOREGULATORY ABILITIES OF HUMAN
BLOOD MONOCYTES CULTURED IN VITRO 1980 6. Størker Jørstad: URAEMIC TOXINS 7. Arne Olav Jenssen: SOME RHEOLOGICAL, CHEMICAL AND STRUCTURAL PROPERTIES
OF MUCOID SPUTUM FROM PATIENTS WITH CHRONIC OBSTRUCTIVE BRONCHITIS 1981 8. Jens Hammerstrøm: CYTOSTATIC AND CYTOLYTIC ACTIVITY OF HUMAN
MONOCYTES AND EFFUSION MACROPHAGES AGAINST TUMOR CELLS IN VITRO 1983 9. Tore Syversen: EFFECTS OF METHYLMERCURY ON RAT BRAIN PROTEIN. 10. Torbjørn Iversen: SQUAMOUS CELL CARCINOMA OF THE VULVA. 1984 11. Tor-Erik Widerøe: ASPECTS OF CONTINUOUS AMBULATORY PERITONEAL DIALYSIS. 12. Anton Hole: ALTERATIONS OF MONOCYTE AND LYMPHOCYTE FUNCTIONS IN
REALTION TO SURGERY UNDER EPIDURAL OR GENERAL ANAESTHESIA. 13. Terje Terjesen: FRACTURE HEALING AN STRESS-PROTECTION AFTER METAL PLATE
FIXATION AND EXTERNAL FIXATION. 14. Carsten Saunte: CLUSTER HEADACHE SYNDROME. 15. Inggard Lereim: TRAFFIC ACCIDENTS AND THEIR CONSEQUENCES. 16. Bjørn Magne Eggen: STUDIES IN CYTOTOXICITY IN HUMAN ADHERENT
MONONUCLEAR BLOOD CELLS. 17. Trond Haug: FACTORS REGULATING BEHAVIORAL EFFECTS OG DRUGS. 1985 18. Sven Erik Gisvold: RESUSCITATION AFTER COMPLETE GLOBAL BRAIN ISCHEMIA. 19. Terje Espevik: THE CYTOSKELETON OF HUMAN MONOCYTES. 20. Lars Bevanger: STUDIES OF THE Ibc (c) PROTEIN ANTIGENS OF GROUP B
STREPTOCOCCI. 21. Ole-Jan Iversen: RETROVIRUS-LIKE PARTICLES IN THE PATHOGENESIS OF PSORIASIS. 22. Lasse Eriksen: EVALUATION AND TREATMENT OF ALCOHOL DEPENDENT
BEHAVIOUR. 23. Per I. Lundmo: ANDROGEN METABOLISM IN THE PROSTATE. 1986 24. Dagfinn Berntzen: ANALYSIS AND MANAGEMENT OF EXPERIMENTAL AND CLINICAL
PAIN. 25. Odd Arnold Kildahl-Andersen: PRODUCTION AND CHARACTERIZATION OF
MONOCYTE-DERIVED CYTOTOXIN AND ITS ROLE IN MONOCYTE-MEDIATED CYTOTOXICITY.
26. Ola Dale: VOLATILE ANAESTHETICS. 1987 27. Per Martin Kleveland: STUDIES ON GASTRIN. 28. Audun N. Øksendal: THE CALCIUM PARADOX AND THE HEART. 29. Vilhjalmur R. Finsen: HIP FRACTURES 1988 30. Rigmor Austgulen: TUMOR NECROSIS FACTOR: A MONOCYTE-DERIVED REGULATOR
OF CELLULAR GROWTH. 31. Tom-Harald Edna: HEAD INJURIES ADMITTED TO HOSPITAL. 32. Joseph D. Borsi: NEW ASPECTS OF THE CLINICAL PHARMACOKINETICS OF
METHOTREXATE.
33. Olav F. M. Sellevold: GLUCOCORTICOIDS IN MYOCARDIAL PROTECTION. 34. Terje Skjærpe: NONINVASIVE QUANTITATION OF GLOBAL PARAMETERS ON LEFT
VENTRICULAR FUNCTION: THE SYSTOLIC PULMONARY ARTERY PRESSURE AND CARDIAC OUTPUT.
35. Eyvind Rødahl: STUDIES OF IMMUNE COMPLEXES AND RETROVIRUS-LIKE ANTIGENS IN PATIENTS WITH ANKYLOSING SPONDYLITIS.
36. Ketil Thorstensen: STUDIES ON THE MECHANISMS OF CELLULAR UPTAKE OF IRON FROM TRANSFERRIN.
37. Anna Midelfart: STUDIES OF THE MECHANISMS OF ION AND FLUID TRANSPORT IN THE BOVINE CORNEA.
38. Eirik Helseth: GROWTH AND PLASMINOGEN ACTIVATOR ACTIVITY OF HUMAN GLIOMAS AND BRAIN METASTASES - WITH SPECIAL REFERENCE TO TRANSFORMING GROWTH FACTOR BETA AND THE EPIDERMAL GROWTH FACTOR RECEPTOR.
39. Petter C. Borchgrevink: MAGNESIUM AND THE ISCHEMIC HEART. 40. Kjell-Arne Rein: THE EFFECT OF EXTRACORPOREAL CIRCULATION ON
SUBCUTANEOUS TRANSCAPILLARY FLUID BALANCE. 41. Arne Kristian Sandvik: RAT GASTRIC HISTAMINE. 42. Carl Bredo Dahl: ANIMAL MODELS IN PSYCHIATRY. 1989 43. Torbjørn A. Fredriksen: CERVICOGENIC HEADACHE. 44. Rolf A. Walstad: CEFTAZIDIME. 45. Rolf Salvesen: THE PUPIL IN CLUSTER HEADACHE. 46. Nils Petter Jørgensen: DRUG EXPOSURE IN EARLY PREGNANCY. 47. Johan C. Ræder: PREMEDICATION AND GENERAL ANAESTHESIA IN OUTPATIENT
GYNECOLOGICAL SURGERY. 48. M. R. Shalaby: IMMUNOREGULATORY PROPERTIES OF TNF-α AND THE RELATED
CYTOKINES. 49. Anders Waage: THE COMPLEX PATTERN OF CYTOKINES IN SEPTIC SHOCK. 50. Bjarne Christian Eriksen: ELECTROSTIMULATION OF THE PELVIC FLOOR IN FEMALE
URINARY INCONTINENCE. 51. Tore B. Halvorsen: PROGNOSTIC FACTORS IN COLORECTAL CANCER. 1990 52. Asbjørn Nordby: CELLULAR TOXICITY OF ROENTGEN CONTRAST MEDIA. 53. Kåre E. Tvedt: X-RAY MICROANALYSIS OF BIOLOGICAL MATERIAL. 54. Tore C. Stiles: COGNITIVE VULNERABILITY FACTORS IN THE DEVELOPMENT AND
MAINTENANCE OF DEPRESSION. 55. Eva Hofsli: TUMOR NECROSIS FACTOR AND MULTIDRUG RESISTANCE. 56. Helge S. Haarstad: TROPHIC EFFECTS OF CHOLECYSTOKININ AND SECRETIN ON THE
RAT PANCREAS. 57. Lars Engebretsen: TREATMENT OF ACUTE ANTERIOR CRUCIATE LIGAMENT INJURIES. 58. Tarjei Rygnestad: DELIBERATE SELF-POISONING IN TRONDHEIM. 59. Arne Z. Henriksen: STUDIES ON CONSERVED ANTIGENIC DOMAINS ON MAJOR OUTER
MEMBRANE PROTEINS FROM ENTEROBACTERIA. 60. Steinar Westin: UNEMPLOYMENT AND HEALTH: Medical and social consequences of a
factory closure in a ten-year controlled follow-up study. 61. Ylva Sahlin: INJURY REGISTRATION, a tool for accident preventive work. 62. Helge Bjørnstad Pettersen: BIOSYNTHESIS OF COMPLEMENT BY HUMAN ALVEOLAR
MACROPHAGES WITH SPECIAL REFERENCE TO SARCOIDOSIS. 63. Berit Schei: TRAPPED IN PAINFUL LOVE. 64. Lars J. Vatten: PROSPECTIVE STUDIES OF THE RISK OF BREAST CANCER IN A
COHORT OF NORWEGIAN WOMAN. 1991 65. Kåre Bergh: APPLICATIONS OF ANTI-C5a SPECIFIC MONOCLONAL ANTIBODIES FOR
THE ASSESSMENT OF COMPLEMENT ACTIVATION. 66. Svein Svenningsen: THE CLINICAL SIGNIFICANCE OF INCREASED FEMORAL
68. Trond Sand: THE EFFECTS OF CLICK POLARITY ON BRAINSTEM AUDITORY EVOKED POTENTIALS AMPLITUDE, DISPERSION, AND LATENCY VARIABLES.
69. Kjetil B. Åsbakk: STUDIES OF A PROTEIN FROM PSORIATIC SCALE, PSO P27, WITH RESPECT TO ITS POTENTIAL ROLE IN IMMUNE REACTIONS IN PSORIASIS.
70. Arnulf Hestnes: STUDIES ON DOWN´S SYNDROME. 71. Randi Nygaard: LONG-TERM SURVIVAL IN CHILDHOOD LEUKEMIA. 72. Bjørn Hagen: THIO-TEPA. 73. Svein Anda: EVALUATION OF THE HIP JOINT BY COMPUTED TOMOGRAMPHY AND
ULTRASONOGRAPHY. 1992 74. Martin Svartberg: AN INVESTIGATION OF PROCESS AND OUTCOME OF SHORT-TERM
PSYCHODYNAMIC PSYCHOTHERAPY. 75. Stig Arild Slørdahl: AORTIC REGURGITATION. 76. Harold C Sexton: STUDIES RELATING TO THE TREATMENT OF SYMPTOMATIC NON-
PSYCHOTIC PATIENTS. 77. Maurice B. Vincent: VASOACTIVE PEPTIDES IN THE OCULAR/FOREHEAD AREA. 78. Terje Johannessen: CONTROLLED TRIALS IN SINGLE SUBJECTS. 79. Turid Nilsen: PYROPHOSPHATE IN HEPATOCYTE IRON METABOLISM. 80. Olav Haraldseth: NMR SPECTROSCOPY OF CEREBRAL ISCHEMIA AND REPERFUSION
IN RAT. 81. Eiliv Brenna: REGULATION OF FUNCTION AND GROWTH OF THE OXYNTIC MUCOSA. 1993 82. Gunnar Bovim: CERVICOGENIC HEADACHE. 83. Jarl Arne Kahn: ASSISTED PROCREATION. 84. Bjørn Naume: IMMUNOREGULATORY EFFECTS OF CYTOKINES ON NK CELLS. 85. Rune Wiseth: AORTIC VALVE REPLACEMENT. 86. Jie Ming Shen: BLOOD FLOW VELOCITY AND RESPIRATORY STUDIES. 87. Piotr Kruszewski: SUNCT SYNDROME WITH SPECIAL REFERENCE TO THE
AUTONOMIC NERVOUS SYSTEM. 88. Mette Haase Moen: ENDOMETRIOSIS. 89. Anne Vik: VASCULAR GAS EMBOLISM DURING AIR INFUSION AND AFTER
DECOMPRESSION IN PIGS. 90. Lars Jacob Stovner: THE CHIARI TYPE I MALFORMATION. 91. Kjell Å. Salvesen: ROUTINE ULTRASONOGRAPHY IN UTERO AND DEVELOPMENT IN
CHILDHOOD. 1994 92. Nina-Beate Liabakk: DEVELOPMENT OF IMMUNOASSAYS FOR TNF AND ITS SOLUBLE
RECEPTORS. 93. Sverre Helge Torp: erbB ONCOGENES IN HUMAN GLIOMAS AND MENINGIOMAS. 94. Olav M. Linaker: MENTAL RETARDATION AND PSYCHIATRY. Past and present. 95. Per Oscar Feet: INCREASED ANTIDEPRESSANT AND ANTIPANIC EFFECT IN
COMBINED TREATMENT WITH DIXYRAZINE AND TRICYCLIC ANTIDEPRESSANTS. 96. Stein Olav Samstad: CROSS SECTIONAL FLOW VELOCITY PROFILES FROM TWO-
DIMENSIONAL DOPPLER ULTRASOUND: Studies on early mitral blood flow. 97. Bjørn Backe: STUDIES IN ANTENATAL CARE. 98. Gerd Inger Ringdal: QUALITY OF LIFE IN CANCER PATIENTS. 99. Torvid Kiserud: THE DUCTUS VENOSUS IN THE HUMAN FETUS. 100. Hans E. Fjøsne: HORMONAL REGULATION OF PROSTATIC METABOLISM. 101. Eylert Brodtkorb: CLINICAL ASPECTS OF EPILEPSY IN THE MENTALLY RETARDED. 102. Roar Juul: PEPTIDERGIC MECHANISMS IN HUMAN SUBARACHNOID HEMORRHAGE. 103. Unni Syversen: CHROMOGRANIN A. Phsysiological and Clinical Role. 1995 104. Odd Gunnar Brakstad: THERMOSTABLE NUCLEASE AND THE nuc GENE IN THE
DIAGNOSIS OF Staphylococcus aureus INFECTIONS. 105. Terje Engan: NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY OF PLASMA
IN MALIGNANT DISEASE. 106. Kirsten Rasmussen: VIOLENCE IN THE MENTALLY DISORDERED. 107. Finn Egil Skjeldestad: INDUCED ABORTION: Timetrends and Determinants. 108. Roar Stenseth: THORACIC EPIDURAL ANALGESIA IN AORTOCORONARY BYPASS
SURGERY.
109. Arild Faxvaag: STUDIES OF IMMUNE CELL FUNCTION in mice infected with MURINE RETROVIRUS.
1996 110. Svend Aakhus: NONINVASIVE COMPUTERIZED ASSESSMENT OF LEFT VENTRICULAR
FUNCTION AND SYSTEMIC ARTERIAL PROPERTIES. Methodology and some clinical applications.
CLAMPING. 113. Sigurd Steinshamn: CYTOKINE MEDIATORS DURING GRANULOCYTOPENIC
INFECTIONS. 114. Hans Stifoss-Hanssen: SEEKING MEANING OR HAPPINESS? 115. Anne Kvikstad: LIFE CHANGE EVENTS AND MARITAL STATUS IN RELATION TO RISK
AND PROGNOSIS OF CANSER. 116. Torbjørn Grøntvedt: TREATMENT OF ACUTE AND CHRONIC ANTERIOR CRUCIATE
LIGAMENT INJURIES. A clinical and biomechanical study. 117. Sigrid Hørven Wigers: CLINICAL STUDIES OF FIBROMYALGIA WITH FOCUS ON
ETIOLOGY, TREATMENT AND OUTCOME. 118. Jan Schjøtt: MYOCARDIAL PROTECTION: Functional and Metabolic Characteristics of Two
Endogenous Protective Principles. 119. Marit Martinussen: STUDIES OF INTESTINAL BLOOD FLOW AND ITS RELATION TO
TRANSITIONAL CIRCULATORY ADAPATION IN NEWBORN INFANTS. 120. Tomm B. Müller: MAGNETIC RESONANCE IMAGING IN FOCAL CEREBRAL ISCHEMIA. 121. Rune Haaverstad: OEDEMA FORMATION OF THE LOWER EXTREMITIES. 122. Magne Børset: THE ROLE OF CYTOKINES IN MULTIPLE MYELOMA, WITH SPECIAL
REFERENCE TO HEPATOCYTE GROWTH FACTOR. 123. Geir Smedslund: A THEORETICAL AND EMPIRICAL INVESTIGATION OF SMOKING,
STRESS AND DISEASE: RESULTS FROM A POPULATION SURVEY. 1997 124. Torstein Vik: GROWTH, MORBIDITY, AND PSYCHOMOTOR DEVELOPMENT IN
INFANTS WHO WERE GROWTH RETARDED IN UTERO. 125. Siri Forsmo: ASPECTS AND CONSEQUENCES OF OPPORTUNISTIC SCREENING FOR
CERVICAL CANCER. Results based on data from three Norwegian counties. 126. Jon S. Skranes: CEREBRAL MRI AND NEURODEVELOPMENTAL OUTCOME IN VERY
LOW BIRTH WEIGHT (VLBW) CHILDREN. A follow-up study of a geographically based year cohort of VLBW children at ages one and six years.
127. Knut Bjørnstad: COMPUTERIZED ECHOCARDIOGRAPHY FOR EVALUTION OF CORONARY ARTERY DISEASE.
128. Grethe Elisabeth Borchgrevink: DIAGNOSIS AND TREATMENT OF WHIPLASH/NECK SPRAIN INJURIES CAUSED BY CAR ACCIDENTS.
129. Tor Elsås: NEUROPEPTIDES AND NITRIC OXIDE SYNTHASE IN OCULAR AUTONOMIC AND SENSORY NERVES.
130. Rolf W. Gråwe: EPIDEMIOLOGICAL AND NEUROPSYCHOLOGICAL PERSPECTIVES ON SCHIZOPHRENIA.
131. Tonje Strømholm: CEREBRAL HAEMODYNAMICS DURING THORACIC AORTIC CROSSCLAMPING. An experimental study in pigs.
1998 132. Martinus Bråten: STUDIES ON SOME PROBLEMS REALTED TO INTRAMEDULLARY
NAILING OF FEMORAL FRACTURES. 133. Ståle Nordgård: PROLIFERATIVE ACTIVITY AND DNA CONTENT AS PROGNOSTIC
INDICATORS IN ADENOID CYSTIC CARCINOMA OF THE HEAD AND NECK. 134. Egil Lien: SOLUBLE RECEPTORS FOR TNF AND LPS: RELEASE PATTERN AND
POSSIBLE SIGNIFICANCE IN DISEASE. 135. Marit Bjørgaas: HYPOGLYCAEMIA IN CHILDREN WITH DIABETES MELLITUS 136. Frank Skorpen: GENETIC AND FUNCTIONAL ANALYSES OF DNA REPAIR IN HUMAN
CELLS. 137. Juan A. Pareja: SUNCT SYNDROME. ON THE CLINICAL PICTURE. ITS DISTINCTION
FROM OTHER, SIMILAR HEADACHES. 138. Anders Angelsen: NEUROENDOCRINE CELLS IN HUMAN PROSTATIC CARCINOMAS
AND THE PROSTATIC COMPLEX OF RAT, GUINEA PIG, CAT AND DOG.
139. Fabio Antonaci: CHRONIC PAROXYSMAL HEMICRANIA AND HEMICRANIA CONTINUA: TWO DIFFERENT ENTITIES?
140. Sven M. Carlsen: ENDOCRINE AND METABOLIC EFFECTS OF METFORMIN WITH SPECIAL EMPHASIS ON CARDIOVASCULAR RISK FACTORES.
1999 141. Terje A. Murberg: DEPRESSIVE SYMPTOMS AND COPING AMONG PATIENTS WITH
CONGESTIVE HEART FAILURE. 142. Harm-Gerd Karl Blaas: THE EMBRYONIC EXAMINATION. Ultrasound studies on the
development of the human embryo. 143. Noèmi Becser Andersen:THE CEPHALIC SENSORY NERVES IN UNILATERAL
HEADACHES. Anatomical background and neurophysiological evaluation. 144. Eli-Janne Fiskerstrand: LASER TREATMENT OF PORT WINE STAINS. A study of the efficacy
and limitations of the pulsed dye laser. Clinical and morfological analyses aimed at improving the therapeutic outcome.
145. Bård Kulseng: A STUDY OF ALGINATE CAPSULE PROPERTIES AND CYTOKINES IN RELATION TO INSULIN DEPENDENT DIABETES MELLITUS.
146. Terje Haug: STRUCTURE AND REGULATION OF THE HUMAN UNG GENE ENCODING URACIL-DNA GLYCOSYLASE.
147. Heidi Brurok: MANGANESE AND THE HEART. A Magic Metal with Diagnostic and Therapeutic Possibilites.
148. Agnes Kathrine Lie: DIAGNOSIS AND PREVALENCE OF HUMAN PAPILLOMAVIRUS INFECTION IN CERVICAL INTRAEPITELIAL NEOPLASIA. Relationship to Cell Cycle Regulatory Proteins and HLA DQBI Genes.
149. Ronald Mårvik: PHARMACOLOGICAL, PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL STUDIES ON ISOLATED STOMACS.
150. Ketil Jarl Holen: THE ROLE OF ULTRASONOGRAPHY IN THE DIAGNOSIS AND TREATMENT OF HIP DYSPLASIA IN NEWBORNS.
151. Irene Hetlevik: THE ROLE OF CLINICAL GUIDELINES IN CARDIOVASCULAR RISK INTERVENTION IN GENERAL PRACTICE.
152. Katarina Tunòn: ULTRASOUND AND PREDICTION OF GESTATIONAL AGE. 153. Johannes Soma: INTERACTION BETWEEN THE LEFT VENTRICLE AND THE SYSTEMIC
ARTERIES. 154. Arild Aamodt: DEVELOPMENT AND PRE-CLINICAL EVALUATION OF A CUSTOM-
MADE FEMORAL STEM. 155. Agnar Tegnander: DIAGNOSIS AND FOLLOW-UP OF CHILDREN WITH SUSPECTED OR
KNOWN HIP DYSPLASIA. 156. Bent Indredavik: STROKE UNIT TREATMENT: SHORT AND LONG-TERM EFFECTS 157. Jolanta Vanagaite Vingen: PHOTOPHOBIA AND PHONOPHOBIA IN PRIMARY
HEADACHES 2000 158. Ola Dalsegg Sæther: PATHOPHYSIOLOGY DURING PROXIMAL AORTIC CROSS-
CLAMPING CLINICAL AND EXPERIMENTAL STUDIES 159. xxxxxxxxx (blind number) 160. Christina Vogt Isaksen: PRENATAL ULTRASOUND AND POSTMORTEM FINDINGS – A
TEN YEAR CORRELATIVE STUDY OF FETUSES AND INFANTS WITH DEVELOPMENTAL ANOMALIES.
161. Holger Seidel: HIGH-DOSE METHOTREXATE THERAPY IN CHILDREN WITH ACUTE LYMPHOCYTIC LEUKEMIA: DOSE, CONCENTRATION, AND EFFECT CONSIDERATIONS.
162. Stein Hallan: IMPLEMENTATION OF MODERN MEDICAL DECISION ANALYSIS INTO CLINICAL DIAGNOSIS AND TREATMENT.
163. Malcolm Sue-Chu: INVASIVE AND NON-INVASIVE STUDIES IN CROSS-COUNTRY SKIERS WITH ASTHMA-LIKE SYMPTOMS.
164. Ole-Lars Brekke: EFFECTS OF ANTIOXIDANTS AND FATTY ACIDS ON TUMOR NECROSIS FACTOR-INDUCED CYTOTOXICITY.
165. Jan Lundbom: AORTOCORONARY BYPASS SURGERY: CLINICAL ASPECTS, COST CONSIDERATIONS AND WORKING ABILITY.
168. Eirik Skogvoll: CARDIAC ARREST Incidence, Intervention and Outcome. 169. Dalius Bansevicius: SHOULDER-NECK REGION IN CERTAIN HEADACHES AND
CHRONIC PAIN SYNDROMES. 170. Bettina Kinge: REFRACTIVE ERRORS AND BIOMETRIC CHANGES AMONG
UNIVERSITY STUDENTS IN NORWAY. 171. Gunnar Qvigstad: CONSEQUENCES OF HYPERGASTRINEMIA IN MAN 172. Hanne Ellekjær: EPIDEMIOLOGICAL STUDIES OF STROKE IN A NORWEGIAN
POPULATION. INCIDENCE, RISK FACTORS AND PROGNOSIS 173. Hilde Grimstad: VIOLENCE AGAINST WOMEN AND PREGNANCY OUTCOME. 174. Astrid Hjelde: SURFACE TENSION AND COMPLEMENT ACTIVATION: Factors influencing
bubble formation and bubble effects after decompression. 175. Kjell A. Kvistad: MR IN BREAST CANCER – A CLINICAL STUDY. 176. Ivar Rossvoll: ELECTIVE ORTHOPAEDIC SURGERY IN A DEFINED POPULATION. Studies
on demand, waiting time for treatment and incapacity for work. 177. Carina Seidel: PROGNOSTIC VALUE AND BIOLOGICAL EFFECTS OF HEPATOCYTE
GROWTH FACTOR AND SYNDECAN-1 IN MULTIPLE MYELOMA. 2001 178. Alexander Wahba: THE INFLUENCE OF CARDIOPULMONARY BYPASS ON PLATELET
FUNCTION AND BLOOD COAGULATION – DETERMINANTS AND CLINICAL CONSEQUENSES
179. Marcus Schmitt-Egenolf: THE RELEVANCE OF THE MAJOR hISTOCOMPATIBILITY COMPLEX FOR THE GENETICS OF PSORIASIS
180. Odrun Arna Gederaas: BIOLOGICAL MECHANISMS INVOLVED IN 5-AMINOLEVULINIC ACID BASED PHOTODYNAMIC THERAPY
181. Pål Richard Romundstad: CANCER INCIDENCE AMONG NORWEGIAN ALUMINIUM WORKERS
182. Henrik Hjorth-Hansen: NOVEL CYTOKINES IN GROWTH CONTROL AND BONE DISEASE OF MULTIPLE MYELOMA
183. Gunnar Morken: SEASONAL VARIATION OF HUMAN MOOD AND BEHAVIOUR 184. Bjørn Olav Haugen: MEASUREMENT OF CARDIAC OUTPUT AND STUDIES OF
VELOCITY PROFILES IN AORTIC AND MITRAL FLOW USING TWO- AND THREE-DIMENSIONAL COLOUR FLOW IMAGING
185. Geir Bråthen: THE CLASSIFICATION AND CLINICAL DIAGNOSIS OF ALCOHOL-RELATED SEIZURES
186. Knut Ivar Aasarød: RENAL INVOLVEMENT IN INFLAMMATORY RHEUMATIC DISEASE. A Study of Renal Disease in Wegener’s Granulomatosis and in Primary Sjögren’s Syndrome
187. Trude Helen Flo: RESEPTORS INVOLVED IN CELL ACTIVATION BY DEFINED URONIC ACID POLYMERS AND BACTERIAL COMPONENTS
188. Bodil Kavli: HUMAN URACIL-DNA GLYCOSYLASES FROM THE UNG GENE: STRUCTRUAL BASIS FOR SUBSTRATE SPECIFICITY AND REPAIR
189. Liv Thommesen: MOLECULAR MECHANISMS INVOLVED IN TNF- AND GASTRIN-MEDIATED GENE REGULATION
190. Turid Lingaas Holmen: SMOKING AND HEALTH IN ADOLESCENCE; THE NORD-TRØNDELAG HEALTH STUDY, 1995-97
191. Øyvind Hjertner: MULTIPLE MYELOMA: INTERACTIONS BETWEEN MALIGNANT PLASMA CELLS AND THE BONE MICROENVIRONMENT
192. Asbjørn Støylen: STRAIN RATE IMAGING OF THE LEFT VENTRICLE BY ULTRASOUND. FEASIBILITY, CLINICAL VALIDATION AND PHYSIOLOGICAL ASPECTS
193. Kristian Midthjell: DIABETES IN ADULTS IN NORD-TRØNDELAG. PUBLIC HEALTH ASPECTS OF DIABETES MELLITUS IN A LARGE, NON-SELECTED NORWEGIAN POPULATION.
194. Guanglin Cui: FUNCTIONAL ASPECTS OF THE ECL CELL IN RODENTS 195. Ulrik Wisløff: CARDIAC EFFECTS OF AEROBIC ENDURANCE TRAINING:
HYPERTROPHY, CONTRACTILITY AND CALCUIM HANDLING IN NORMAL AND FAILING HEART
196. Øyvind Halaas: MECHANISMS OF IMMUNOMODULATION AND CELL-MEDIATED CYTOTOXICITY INDUCED BY BACTERIAL PRODUCTS
197. Tore Amundsen: PERFUSION MR IMAGING IN THE DIAGNOSIS OF PULMONARY EMBOLISM
198. Nanna Kurtze: THE SIGNIFICANCE OF ANXIETY AND DEPRESSION IN FATIQUE AND PATTERNS OF PAIN AMONG INDIVIDUALS DIAGNOSED WITH FIBROMYALGIA: RELATIONS WITH QUALITY OF LIFE, FUNCTIONAL DISABILITY, LIFESTYLE, EMPLOYMENT STATUS, CO-MORBIDITY AND GENDER
199. Tom Ivar Lund Nilsen: PROSPECTIVE STUDIES OF CANCER RISK IN NORD-TRØNDELAG: THE HUNT STUDY. Associations with anthropometric, socioeconomic, and lifestyle risk factors
200. Asta Kristine Håberg: A NEW APPROACH TO THE STUDY OF MIDDLE CEREBRAL ARTERY OCCLUSION IN THE RAT USING MAGNETIC RESONANCE TECHNIQUES
2002 201. Knut Jørgen Arntzen: PREGNANCY AND CYTOKINES 202. Henrik Døllner: INFLAMMATORY MEDIATORS IN PERINATAL INFECTIONS 203. Asta Bye: LOW FAT, LOW LACTOSE DIET USED AS PROPHYLACTIC TREATMENT OF
ACUTE INTESTINAL REACTIONS DURING PELVIC RADIOTHERAPY. A PROSPECTIVE RANDOMISED STUDY.
204. Sylvester Moyo: STUDIES ON STREPTOCOCCUS AGALACTIAE (GROUP B STREPTOCOCCUS) SURFACE-ANCHORED MARKERS WITH EMPHASIS ON STRAINS AND HUMAN SERA FROM ZIMBABWE.
205. Knut Hagen: HEAD-HUNT: THE EPIDEMIOLOGY OF HEADACHE IN NORD-TRØNDELAG 206. Li Lixin: ON THE REGULATION AND ROLE OF UNCOUPLING PROTEIN-2 IN INSULIN
PRODUCING ß-CELLS 207. Anne Hildur Henriksen: SYMPTOMS OF ALLERGY AND ASTHMA VERSUS MARKERS OF
LOWER AIRWAY INFLAMMATION AMONG ADOLESCENTS 208. Egil Andreas Fors: NON-MALIGNANT PAIN IN RELATION TO PSYCHOLOGICAL AND
ENVIRONTENTAL FACTORS. EXPERIENTAL AND CLINICAL STUDES OF PAIN WITH FOCUS ON FIBROMYALGIA
209. Pål Klepstad: MORPHINE FOR CANCER PAIN 210. Ingunn Bakke: MECHANISMS AND CONSEQUENCES OF PEROXISOME PROLIFERATOR-
INDUCED HYPERFUNCTION OF THE RAT GASTRIN PRODUCING CELL 211. Ingrid Susann Gribbestad: MAGNETIC RESONANCE IMAGING AND SPECTROSCOPY OF
BREAST CANCER 212. Rønnaug Astri Ødegård: PREECLAMPSIA – MATERNAL RISK FACTORS AND FETAL
GROWTH 213. Johan Haux: STUDIES ON CYTOTOXICITY INDUCED BY HUMAN NATURAL KILLER
CELLS AND DIGITOXIN 214. Turid Suzanne Berg-Nielsen: PARENTING PRACTICES AND MENTALLY DISORDERED
ADOLESCENTS 215. Astrid Rydning: BLOOD FLOW AS A PROTECTIVE FACTOR FOR THE STOMACH
MUCOSA. AN EXPERIMENTAL STUDY ON THE ROLE OF MAST CELLS AND SENSORY AFFERENT NEURONS
2003 216. Jan Pål Loennechen: HEART FAILURE AFTER MYOCARDIAL INFARCTION. Regional
Differences, Myocyte Function, Gene Expression, and Response to Cariporide, Losartan, and Exercise Training.
217. Elisabeth Qvigstad: EFFECTS OF FATTY ACIDS AND OVER-STIMULATION ON INSULIN SECRETION IN MAN
218. Arne Åsberg: EPIDEMIOLOGICAL STUDIES IN HEREDITARY HEMOCHROMATOSIS: PREVALENCE, MORBIDITY AND BENEFIT OF SCREENING.
219. Johan Fredrik Skomsvoll: REPRODUCTIVE OUTCOME IN WOMEN WITH RHEUMATIC DISEASE. A population registry based study of the effects of inflammatory rheumatic disease and connective tissue disease on reproductive outcome in Norwegian women in 1967-1995.
220. Siv Mørkved: URINARY INCONTINENCE DURING PREGNANCY AND AFTER DELIVERY: EFFECT OF PELVIC FLOOR MUSCLE TRAINING IN PREVENTION AND TREATMENT
221. Marit S. Jordhøy: THE IMPACT OF COMPREHENSIVE PALLIATIVE CARE 222. Tom Christian Martinsen: HYPERGASTRINEMIA AND HYPOACIDITY IN RODENTS –
CAUSES AND CONSEQUENCES 223. Solveig Tingulstad: CENTRALIZATION OF PRIMARY SURGERY FOR OVARAIN CANCER.
FEASIBILITY AND IMPACT ON SURVIVAL
224. Haytham Eloqayli: METABOLIC CHANGES IN THE BRAIN CAUSED BY EPILEPTIC SEIZURES
225. Torunn Bruland: STUDIES OF EARLY RETROVIRUS-HOST INTERACTIONS – VIRAL DETERMINANTS FOR PATHOGENESIS AND THE INFLUENCE OF SEX ON THE SUSCEPTIBILITY TO FRIEND MURINE LEUKAEMIA VIRUS INFECTION
226. Torstein Hole: DOPPLER ECHOCARDIOGRAPHIC EVALUATION OF LEFT VENTRICULAR FUNCTION IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION
227. Vibeke Nossum: THE EFFECT OF VASCULAR BUBBLES ON ENDOTHELIAL FUNCTION 228. Sigurd Fasting: ROUTINE BASED RECORDING OF ADVERSE EVENTS DURING
ANAESTHESIA – APPLICATION IN QUALITY IMPROVEMENT AND SAFETY 229. Solfrid Romundstad: EPIDEMIOLOGICAL STUDIES OF MICROALBUMINURIA. THE
NORD-TRØNDELAG HEALTH STUDY 1995-97 (HUNT 2) 230. Geir Torheim: PROCESSING OF DYNAMIC DATA SETS IN MAGNETIC RESONANCE
IMAGING 231. Catrine Ahlén: SKIN INFECTIONS IN OCCUPATIONAL SATURATION DIVERS IN THE
NORTH SEA AND THE IMPACT OF THE ENVIRONMENT 232. Arnulf Langhammer: RESPIRATORY SYMPTOMS, LUNG FUNCTION AND BONE
MINERAL DENSITY IN A COMPREHENSIVE POPULATION SURVEY. THE NORD-TRØNDELAG HEALTH STUDY 1995-97. THE BRONCHIAL OBSTRUCTION IN NORD-TRØNDELAG STUDY
233. Einar Kjelsås: EATING DISORDERS AND PHYSICAL ACTIVITY IN NON-CLINICAL SAMPLES
234. Arne Wibe: RECTAL CANCER TREATMENT IN NORWAY – STANDARDISATION OF SURGERY AND QUALITY ASSURANCE
2004 235. Eivind Witsø: BONE GRAFT AS AN ANTIBIOTIC CARRIER 236. Anne Mari Sund: DEVELOPMENT OF DEPRESSIVE SYMPTOMS IN EARLY
ADOLESCENCE 237. Hallvard Lærum: EVALUATION OF ELECTRONIC MEDICAL RECORDS – A CLINICAL
TASK PERSPECTIVE 238. Gustav Mikkelsen: ACCESSIBILITY OF INFORMATION IN ELECTRONIC PATIENT
RECORDS; AN EVALUATION OF THE ROLE OF DATA QUALITY 239. Steinar Krokstad: SOCIOECONOMIC INEQUALITIES IN HEALTH AND DISABILITY.
SOCIAL EPIDEMIOLOGY IN THE NORD-TRØNDELAG HEALTH STUDY (HUNT), NORWAY
240. Arne Kristian Myhre: NORMAL VARIATION IN ANOGENITAL ANATOMY AND MICROBIOLOGY IN NON-ABUSED PRESCHOOL CHILDREN
241. Ingunn Dybedal: NEGATIVE REGULATORS OF HEMATOPOIETEC STEM AND PROGENITOR CELLS
242. Beate Sitter: TISSUE CHARACTERIZATION BY HIGH RESOLUTION MAGIC ANGLE SPINNING MR SPECTROSCOPY
243. Per Arne Aas: MACROMOLECULAR MAINTENANCE IN HUMAN CELLS – REPAIR OF URACIL IN DNA AND METHYLATIONS IN DNA AND RNA
244. Anna Bofin: FINE NEEDLE ASPIRATION CYTOLOGY IN THE PRIMARY INVESTIGATION OF BREAST TUMOURS AND IN THE DETERMINATION OF TREATMENT STRATEGIES
245. Jim Aage Nøttestad: DEINSTITUTIONALIZATION AND MENTAL HEALTH CHANGES AMONG PEOPLE WITH MENTAL RETARDATION
246. Reidar Fossmark: GASTRIC CANCER IN JAPANESE COTTON RATS 247. Wibeke Nordhøy: MANGANESE AND THE HEART, INTRACELLULAR MR RELAXATION
AND WATER EXCHANGE ACROSS THE CARDIAC CELL MEMBRANE 2005 248. Sturla Molden: QUANTITATIVE ANALYSES OF SINGLE UNITS RECORDED FROM THE
HIPPOCAMPUS AND ENTORHINAL CORTEX OF BEHAVING RATS 249. Wenche Brenne Drøyvold: EPIDEMIOLOGICAL STUDIES ON WEIGHT CHANGE AND
HEALTH IN A LARGE POPULATION. THE NORD-TRØNDELAG HEALTH STUDY (HUNT)
250. Ragnhild Støen: ENDOTHELIUM-DEPENDENT VASODILATION IN THE FEMORAL ARTERY OF DEVELOPING PIGLETS
251. Aslak Steinsbekk: HOMEOPATHY IN THE PREVENTION OF UPPER RESPIRATORY TRACT INFECTIONS IN CHILDREN
252. Hill-Aina Steffenach: MEMORY IN HIPPOCAMPAL AND CORTICO-HIPPOCAMPAL CIRCUITS
253. Eystein Stordal: ASPECTS OF THE EPIDEMIOLOGY OF DEPRESSIONS BASED ON SELF-RATING IN A LARGE GENERAL HEALTH STUDY (THE HUNT-2 STUDY)
254. Viggo Pettersen: FROM MUSCLES TO SINGING: THE ACTIVITY OF ACCESSORY BREATHING MUSCLES AND THORAX MOVEMENT IN CLASSICAL SINGING
255. Marianne Fyhn: SPATIAL MAPS IN THE HIPPOCAMPUS AND ENTORHINAL CORTEX 256. Robert Valderhaug: OBSESSIVE-COMPULSIVE DISORDER AMONG CHILDREN AND
ADOLESCENTS: CHARACTERISTICS AND PSYCHOLOGICAL MANAGEMENT OF PATIENTS IN OUTPATIENT PSYCHIATRIC CLINICS
257. Erik Skaaheim Haug: INFRARENAL ABDOMINAL AORTIC ANEURYSMS – COMORBIDITY AND RESULTS FOLLOWING OPEN SURGERY
258. Daniel Kondziella: GLIAL-NEURONAL INTERACTIONS IN EXPERIMENTAL BRAIN DISORDERS
259. Vegard Heimly Brun: ROUTES TO SPATIAL MEMORY IN HIPPOCAMPAL PLACE CELLS 260. Kenneth McMillan: PHYSIOLOGICAL ASSESSMENT AND TRAINING OF ENDURANCE
AND STRENGTH IN PROFESSIONAL YOUTH SOCCER PLAYERS 261. Marit Sæbø Indredavik: MENTAL HEALTH AND CEREBRAL MAGNETIC RESONANCE
IMAGING IN ADOLESCENTS WITH LOW BIRTH WEIGHT 262. Ole Johan Kemi: ON THE CELLULAR BASIS OF AEROBIC FITNESS, INTENSITY-
DEPENDENCE AND TIME-COURSE OF CARDIOMYOCYTE AND ENDOTHELIAL ADAPTATIONS TO EXERCISE TRAINING
264. Hild Fjærtoft: EXTENDED STROKE UNIT SERVICE AND EARLY SUPPORTED DISCHARGE. SHORT AND LONG-TERM EFFECTS
265. Grete Dyb: POSTTRAUMATIC STRESS REACTIONS IN CHILDREN AND ADOLESCENTS 266. Vidar Fykse: SOMATOSTATIN AND THE STOMACH 267. Kirsti Berg: OXIDATIVE STRESS AND THE ISCHEMIC HEART: A STUDY IN PATIENTS
UNDERGOING CORONARY REVASCULARIZATION 268. Björn Inge Gustafsson: THE SEROTONIN PRODUCING ENTEROCHROMAFFIN CELL,
AND EFFECTS OF HYPERSEROTONINEMIA ON HEART AND BONE 2006 269. Torstein Baade Rø: EFFECTS OF BONE MORPHOGENETIC PROTEINS, HEPATOCYTE
GROWTH FACTOR AND INTERLEUKIN-21 IN MULTIPLE MYELOMA 270. May-Britt Tessem: METABOLIC EFFECTS OF ULTRAVIOLET RADIATION ON THE
ANTERIOR PART OF THE EYE 271. Anne-Sofie Helvik: COPING AND EVERYDAY LIFE IN A POPULATION OF ADULTS
WITH HEARING IMPAIRMENT 272. Therese Standal: MULTIPLE MYELOMA: THE INTERPLAY BETWEEN MALIGNANT
PLASMA CELLS AND THE BONE MARROW MICROENVIRONMENT 273. Ingvild Saltvedt: TREATMENT OF ACUTELY SICK, FRAIL ELDERLY PATIENTS IN A
GERIATRIC EVALUATION AND MANAGEMENT UNIT – RESULTS FROM A PROSPECTIVE RANDOMISED TRIAL
274. Birger Henning Endreseth: STRATEGIES IN RECTAL CANCER TREATMENT – FOCUS ON EARLY RECTAL CANCER AND THE INFLUENCE OF AGE ON PROGNOSIS
275. Anne Mari Aukan Rokstad: ALGINATE CAPSULES AS BIOREACTORS FOR CELL THERAPY
276. Mansour Akbari: HUMAN BASE EXCISION REPAIR FOR PRESERVATION OF GENOMIC STABILITY
277. Stein Sundstrøm: IMPROVING TREATMENT IN PATIENTS WITH LUNG CANCER – RESULTS FROM TWO MULITCENTRE RANDOMISED STUDIES
278. Hilde Pleym: BLEEDING AFTER CORONARY ARTERY BYPASS SURGERY - STUDIES ON HEMOSTATIC MECHANISMS, PROPHYLACTIC DRUG TREATMENT AND EFFECTS OF AUTOTRANSFUSION
279. Line Merethe Oldervoll: PHYSICAL ACTIVITY AND EXERCISE INTERVENTIONS IN CANCER PATIENTS
280. Boye Welde: THE SIGNIFICANCE OF ENDURANCE TRAINING, RESISTANCE TRAINING AND MOTIVATIONAL STYLES IN ATHLETIC PERFORMANCE AMONG ELITE JUNIOR CROSS-COUNTRY SKIERS
281. Per Olav Vandvik: IRRITABLE BOWEL SYNDROME IN NORWAY, STUDIES OF PREVALENCE, DIAGNOSIS AND CHARACTERISTICS IN GENERAL PRACTICE AND IN THE POPULATION
282. Idar Kirkeby-Garstad: CLINICAL PHYSIOLOGY OF EARLY MOBILIZATION AFTER CARDIAC SURGERY
283. Linn Getz: SUSTAINABLE AND RESPONSIBLE PREVENTIVE MEDICINE. CONCEPTUALISING ETHICAL DILEMMAS ARISING FROM CLINICAL IMPLEMENTATION OF ADVANCING MEDICAL TECHNOLOGY
284. Eva Tegnander: DETECTION OF CONGENITAL HEART DEFECTS IN A NON-SELECTED POPULATION OF 42,381 FETUSES
285. Kristin Gabestad Nørsett: GENE EXPRESSION STUDIES IN GASTROINTESTINAL PATHOPHYSIOLOGY AND NEOPLASIA
286. Per Magnus Haram: GENETIC VS. AQUIRED FITNESS: METABOLIC, VASCULAR AND CARDIOMYOCYTE ADAPTATIONS
287. Agneta Johansson: GENERAL RISK FACTORS FOR GAMBLING PROBLEMS AND THE PREVALENCE OG PATHOLOGICAL GAMBLING IN NORWAY
288. Svein Artur Jensen: THE PREVALENCE OF SYMPTOMATIC ARTERIAL DISEASE OF THE LOWER LIMB
289. Charlotte Björk Ingul: QUANITIFICATION OF REGIONAL MYOCARDIAL FUNCTION BY STRAIN RATE AND STRAIN FOR EVALUATION OF CORONARY ARTERY DISEASE. AUTOMATED VERSUS MANUAL ANALYSIS DURING ACUTE MYOCARDIAL INFARCTION AND DOBUTAMINE STRESS ECHOCARDIOGRAPHY
290. Jakob Nakling: RESULTS AND CONSEQUENCES OF ROUTINE ULTRASOUND SCREENING IN PREGNANCY – A GEOGRAPHIC BASED POPULATION STUDY
291. Anne Engum: DEPRESSION AND ANXIETY – THEIR RELATIONS TO THYROID DYSFUNCTION AND DIABETES IN A LARGE EPIDEMIOLOGICAL STUDY
292. Ottar Bjerkeset: ANXIETY AND DEPRESSION IN THE GENERAL POPULATION: RISK FACTORS, INTERVENTION AND OUTCOME – THE NORD-TRØNDELAG HEALTH STUDY (HUNT)
293. Jon Olav Drogset: RESULTS AFTER SURGICAL TREATMENT OF ANTERIOR CRUCIATE LIGAMENT INJURIES – A CLINICAL STUDY
294. Lars Fosse: MECHANICAL BEHAVIOUR OF COMPACTED MORSELLISED BONE – AN EXPERIMENTAL IN VITRO STUDY
295. Gunilla Klensmeden Fosse: MENTAL HEALTH OF PSYCHIATRIC OUTPATIENTS BULLIED IN CHILDHOOD
296. Paul Jarle Mork: MUSCLE ACTIVITY IN WORK AND LEISURE AND ITS ASSOCIATION TO MUSCULOSKELETAL PAIN
297. Björn Stenström: LESSONS FROM RODENTS: I: MECHANISMS OF OBESITY SURGERY – ROLE OF STOMACH. II: CARCINOGENIC EFFECTS OF HELICOBACTER PYLORI AND SNUS IN THE STOMACH
298. Haakon R. Skogseth: INVASIVE PROPERTIES OF CANCER – A TREATMENT TARGET? IN VITRO STUDIES IN HUMAN PROSTATE CANCER CELL LINES
299. Janniche Hammer: GLUTAMATE METABOLISM AND CYCLING IN MESIAL TEMPORAL LOBE EPILEPSY
300. May Britt Drugli: YOUNG CHILDREN TREATED BECAUSE OF ODD/CD: CONDUCT PROBLEMS AND SOCIAL COMPETENCIES IN DAY-CARE AND SCHOOL SETTINGS
301. Arne Skjold: MAGNETIC RESONANCE KINETICS OF MANGANESE DIPYRIDOXYL DIPHOSPHATE (MnDPDP) IN HUMAN MYOCARDIUM. STUDIES IN HEALTHY VOLUNTEERS AND IN PATIENTS WITH RECENT MYOCARDIAL INFARCTION
302. Siri Malm: LEFT VENTRICULAR SYSTOLIC FUNCTION AND MYOCARDIAL PERFUSION ASSESSED BY CONTRAST ECHOCARDIOGRAPHY
303. Valentina Maria do Rosario Cabral Iversen: MENTAL HEALTH AND PSYCHOLOGICAL ADAPTATION OF CLINICAL AND NON-CLINICAL MIGRANT GROUPS
304. Lasse Løvstakken: SIGNAL PROCESSING IN DIAGNOSTIC ULTRASOUND: ALGORITHMS FOR REAL-TIME ESTIMATION AND VISUALIZATION OF BLOOD FLOW VELOCITY
305. Elisabeth Olstad: GLUTAMATE AND GABA: MAJOR PLAYERS IN NEURONAL METABOLISM
306. Lilian Leistad: THE ROLE OF CYTOKINES AND PHOSPHOLIPASE A2s IN ARTICULAR CARTILAGE CHONDROCYTES IN RHEUMATOID ARTHRITIS AND OSTEOARTHRITIS
307. Arne Vaaler: EFFECTS OF PSYCHIATRIC INTENSIVE CARE UNIT IN AN ACUTE PSYCIATHRIC WARD